WO2023173272A1 - 靶向gprc5d的全人源嵌合抗原受体(car)及其应用 - Google Patents

靶向gprc5d的全人源嵌合抗原受体(car)及其应用 Download PDF

Info

Publication number
WO2023173272A1
WO2023173272A1 PCT/CN2022/080836 CN2022080836W WO2023173272A1 WO 2023173272 A1 WO2023173272 A1 WO 2023173272A1 CN 2022080836 W CN2022080836 W CN 2022080836W WO 2023173272 A1 WO2023173272 A1 WO 2023173272A1
Authority
WO
WIPO (PCT)
Prior art keywords
car
cells
seq
gprc5d
amino acid
Prior art date
Application number
PCT/CN2022/080836
Other languages
English (en)
French (fr)
Inventor
谭涛超
戴振宇
骆倩
赵雅
张艳英
穆伟
魏巧娥
刘建伟
金亮
Original Assignee
上海驯鹿生物技术有限公司
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by 上海驯鹿生物技术有限公司 filed Critical 上海驯鹿生物技术有限公司
Priority to PCT/CN2022/080836 priority Critical patent/WO2023173272A1/zh
Priority to CA3241997A priority patent/CA3241997A1/en
Priority to IL313798A priority patent/IL313798A/en
Priority to KR1020247024554A priority patent/KR20240125026A/ko
Priority to PCT/CN2022/140769 priority patent/WO2023116782A1/zh
Priority to CN202280083287.1A priority patent/CN118451177A/zh
Priority to AU2022418639A priority patent/AU2022418639A1/en
Publication of WO2023173272A1 publication Critical patent/WO2023173272A1/zh

Links

Images

Classifications

    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K39/395—Antibodies; Immunoglobulins; Immune serum, e.g. antilymphocytic serum
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/705—Receptors; Cell surface antigens; Cell surface determinants
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • C07K16/18—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans
    • C07K16/28—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants

Definitions

  • This application relates to the field of biomedicine, specifically to CARs targeting GPRC5D and host cells expressing CARs targeting GPRC5D, as well as their preparation methods and applications.
  • Multiple myeloma is a plasma cell tumor that occurs in the bone marrow.
  • the tumor causes conditions such as hypercalcemia, anemia, renal dysfunction, osteonecrosis, and bone marrow failure.
  • Multiple myeloma is currently the second most common blood cancer [1] .
  • WHO World Health Organization
  • the incidence and mortality rates of the Asian population are 36% and 42% respectively, ranking first among all continents.
  • the global incidence and mortality ratio of multiple myeloma is 1.8:1.1, and that in Asia is 1.1:0.76. It can be seen that the survival rate of multiple myeloma in the affected population is low.
  • the first main reason is that the median age of the affected people is relatively high at about 66 years old, and the incidence rate among people under 40 years old is about 2%.
  • Targeted therapy product targets for multiple myeloma mainly include BCMA, CD38, CD138, GPRC5D, etc. [2] .
  • CD38 and CD138 are expressed on cells in normal tissues and on hematopoietic stem cells. Targeted therapy has greater side effects after removal, often causing damage to normal organs or damaging the autoimmune system.
  • BCMA and GPRC5D are mainly expressed in plasma cells. Or on plasma cells in myeloma, which can be compensated by the continuous regeneration of the body's own B cells.
  • BCMA target products mainly include REGN5458 developed by Regeneron Pharmaceuticals, Inc. (REGN), Teclistama developed by Johnson & Johnson, and AMG420 by Amgen. REGN5458 has currently completed Phase I clinical trials (NCT03761108).
  • bb2121 a CAR-T product developed by Bristol-Myers Squibb (BMS) targeting the BCMA target of multiple myeloma, was approved by the FDA in May 2021 as the first CAR-T product to be marketed for multiple myeloma.
  • the overall response rate (ORR) of this product is 72%, and 28% of patients can achieve stringent complete response (sCR) [4] .
  • sCR stringent complete response
  • this product brings hope for the cure of multiple bone marrow, according to the published data, the 22-month progression-free survival rate of patients with complete response (CR) is less than 50%, which shows that the treatment is late. There is also a higher rate of recurrence.
  • GPRC5D is more specific than BCMA. It is only expressed in plasma cells of myeloma patients, while it is almost not expressed in normal tissues. Obvious RNA and protein expression can only be detected in hair follicle tissue [5], [6] . Comparing the phenotypes of GPRC5D knockout mice and normal wild-type mice (including body weight, organ morphology differences, reproductive rate, etc.), no significant differences were found [7] . It can be seen that GPRC5D deletion has an impact on survival and normal organ growth and development. Metabolism is not required and clearance has minor side effects.
  • BCMA expression is low in patients in the late stage of BCMA CAR-T therapy, and GPRC5D can also be targeted for treatment.
  • the GPRC5D/CD3 dual-antibody Talquemab developed by Johnson & Johnson is also in phase 1/2 clinical studies.
  • the response rate at the medium dose is 70%, of which 65% Triple drug-resistant patients responded, and 83% of quintuple drug-resistant patients responded [6] .
  • the main CAR-T products include MCARH109 developed by Eureka, which is in phase I clinical trials. Data reported at the 2021 ASH Annual Meeting showed that among 12 patients who received GPRC5D CAR-T injection, 3/12 had PR partial response, and 3/ 12 were VGPR very good partial response, and 2/12 were sCR strict complete response (sCR) [5] .
  • chimeric antigen receptors whose extracellular antigen-binding domains include one or more antibody molecules targeting GPRC5D, or antigen-binding fragments thereof, whose heavy chain variable
  • the HCDR1, HCDR2 and HCDR3 of the area are selected from one of the following combinations:
  • HCDR1 The sequence of HCDR1 is GGSFSGYY (SEQ ID NO: 1);
  • HCDR2 The sequence of HCDR2 is INHSGST (SEQ ID NO: 2);
  • HCDR3 is ARARRYGGRTRFDP (SEQ ID NO: 3);
  • HCDR1 is GFIFSSYG (SEQ ID NO: 4);
  • HCDR2 The sequence of HCDR2 is ISSSGDYT (SEQ ID NO: 5);
  • HCDR3 The sequence of HCDR3 is ARMSFRRYDH (SEQ ID NO: 6);
  • HCDR1 is GFSFSGYI (SEQ ID NO: 7);
  • HCDR2 The sequence of HCDR2 is TSSSGTET (SEQ ID NO: 8);
  • HCDR3 is ARYYSKYGRSYHVDS (SEQ ID NO: 9).
  • the extracellular antigen-binding domain includes a plurality (such as two) of the antibody molecules or antigen-binding fragments thereof connected in series.
  • the antibody molecules or antigen-binding fragments thereof are connected by a connecting peptide. connected.
  • the two said antibody molecules or antigen-binding fragments thereof are the same or different.
  • the antibody molecule is a single domain antibody.
  • the antibody molecule is a fully human single domain antibody.
  • the heavy chain variable region (or the single domain antibody) includes the amino acid sequence set forth in SEQ ID NO: 10, 11, or 12; or the heavy chain variable region includes the same amino acid sequence as SEQ ID NO. :
  • the sequence shown in 10, 11 or 12 has an amino acid sequence of at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity and is capable of specificity Sexually binding GPRC5D.
  • the CAR includes the extracellular antigen-binding domain, the transmembrane domain and the intracellular signaling domain in order from the N-terminus to the C-terminus.
  • the CAR further includes a signal peptide at the N-terminus.
  • the signal peptide includes the amino acid sequence set forth in SEQ ID NO: 17.
  • the CAR further includes a hinge region between the extracellular antigen binding domain and the transmembrane domain.
  • the hinge region includes the amino acid sequence set forth in SEQ ID NO: 19.
  • the transmembrane region includes the amino acid sequence set forth in SEQ ID NO: 21.
  • the intracellular signaling domain includes a 4-1BB intracellular domain and a CD3 ⁇ intracellular domain.
  • the 4-1BB intracellular domain includes the amino acid sequence set forth in SEQ ID NO: 23.
  • the CD3 ⁇ intracellular domain includes the amino acid sequence set forth in SEQ ID NO: 25.
  • the end of the CAR is also connected with a safety switch for CAR-T cell clearance.
  • a safety switch is attached to the C-terminus of the CAR.
  • the safety switch includes: tEGFR, or a fusion protein with suicide capability.
  • the CAR has a truncated form of the EGFR molecule (tEGFR) attached to its C-terminus.
  • tEGFR truncated form of the EGFR molecule
  • a CSF2RA signal peptide is also linked to the N-terminus of tEGFR.
  • the tEGFR includes the amino acid sequence set forth in SEQ ID NO: 31.
  • the CSF2RA signal peptide includes the amino acid sequence set forth in SEQ ID NO: 29.
  • the safety switch is linked to the CAR via a self-cleaving polypeptide.
  • the self-cleaving polypeptide includes the amino acid sequence set forth in SEQ ID NO: 27 or 33.
  • the CAR has the amino acid sequence shown in SEQ ID NO: 35, 37 or 39; or the amino acid sequence of the CAR is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity.
  • bispecific chimeric antigen receptors whose extracellular antigen-binding domains include (i) an antibody molecule targeting GPRC5D or an antigen-binding fragment thereof and (ii) targeting a second target Dot antibody molecules or antigen-binding fragments thereof, the single domain antibody heavy chain variable region (V HH ) and full anti-scFv (variable region) of the GPRC5D-targeting antibody molecule are as described above, and the second antibody molecule Selected from CD3, BCMA or combinations thereof.
  • CARs bispecific chimeric antigen receptors
  • this article also provides isolated nucleic acid molecules encoding the above-mentioned CAR.
  • the nucleic acid molecule includes any one of SEQ ID NOs: 13, 14, 15, 16, 18, 20, 22, 24, 26, 28, 30, 32, 34, 36 and 38. Nucleotide sequence.
  • this article also provides expression vectors, which include the above-mentioned nucleic acid molecules.
  • the expression vector is selected from the group consisting of plasmids, retroviral vectors, and lentiviral vectors.
  • this article also provides host cells, which include the above-mentioned CAR, nucleic acid molecules or expression vectors.
  • the host cells are immune cells.
  • the host cells include T cells or NK cells, preferably 293T cells.
  • this article also provides engineered immune cells expressing the above-mentioned CAR.
  • the engineered immune cells include immune cells, preferably T cells or NK cells.
  • the engineered immune cells include autologous or allogeneic cells.
  • this article also provides a pharmaceutical composition, which includes the above-mentioned cells and a pharmaceutically acceptable carrier.
  • this article also provides the use of the above-mentioned CAR, nucleic acid molecules, expression vectors, host cells, or engineered immune cells in preparing drugs for preventing or treating GPRC5D-related diseases.
  • the GPRC5D-related disease is a cancer or an autoimmune disease, preferably a plasma cell malignant disease, such as multiple myeloma; or a B cell malignant disease, such as Hodgkin's lymphoma or non-Hodgkin lymphoma. gold lymphoma.
  • a plasma cell malignant disease such as multiple myeloma
  • a B cell malignant disease such as Hodgkin's lymphoma or non-Hodgkin lymphoma. gold lymphoma.
  • this article also provides methods for preventing or treating GPRC5D-related diseases, including administering a therapeutically effective amount of the above-mentioned engineered immune cells or pharmaceutical composition to a subject in need.
  • the method further comprises administering an EGFR antibody to the subject to inhibit the activity of the engineered immune cells or the pharmaceutical composition.
  • the GPRC5D-related disease is a cancer or an autoimmune disease, preferably a plasma cell malignant disease, such as multiple myeloma; or a B cell malignant disease, such as Hodgkin's lymphoma or non-Hodgkin lymphoma. gold lymphoma.
  • a plasma cell malignant disease such as multiple myeloma
  • a B cell malignant disease such as Hodgkin's lymphoma or non-Hodgkin lymphoma. gold lymphoma.
  • this application Based on the extremely specific expression profile of GPRC5D, its potential safety and the good efficacy of BCMA therapy, this application has developed a CAR-T product targeting GPRC5D for the treatment of refractory relapsed multiple myeloma.
  • the development of the GPRC5D binding domain comes from the screening of humanized single domain antibody libraries. It has a relatively simple structure and low immunogenicity.
  • FIG. 1 shows the structural schematic diagram of GPRC5D CAR vector construction.
  • CAR includes extracellular signal peptide (SP), ScFv (VL-linker-VH) or Single domain (V HH ) in the binding region of the target antigen GPRC5D, hinge region and transmembrane region (CD8a) between the cell membrane and extracellular binding region.
  • hinge+TM 4-1BB costimulatory molecule
  • CD3 ⁇ intracellular domain CD3 ⁇ intracellular domain
  • T2A self-cleaving peptide truncated tEGFR.
  • Figure 2 shows the expression of tEGFR in CAR-T cells in each group.
  • Figures 3A-3C show the killing results of various groups of CAR-T cells against various target cells (8226-luc, U266-luc, MM1.s-luc, CCRF-luc, and Raji-luc).
  • Figure 4 shows the in vitro proliferation of various CAR-T cells after multiple stimulations of target cells (8226).
  • Figure 5 shows the results of CD107a degranulation of CAR-T cells in each group by tumor cells derived from different tissues.
  • Antibodies refer to immunoglobulins secreted by plasma cells (effector B cells) and used by the body's immune system to neutralize foreign substances (polypeptides, viruses, bacteria, etc.). This foreign substance is accordingly called an antigen.
  • the basic structure of a classic antibody molecule is a tetramer composed of two identical heavy chains and two identical light chains. According to the conservative differences in amino acid sequences, heavy and light chains are divided into variable regions (V) located at the amino terminus and constant regions (C) located at the carboxyl terminus. The variable regions of a heavy chain and a light chain interact to form the antigen-binding site (Fv).
  • variable region the composition and order of amino acid residues in certain regions are more variable than other regions (framework regions, FR) within the variable region, which are called hypervariable regions (HVR).
  • the hypervariable regions are actually antibodies. Key site for antigen binding. Because these hypervariable region sequences are complementary to antigenic determinants, they are also called complementarity-determining regions (CDRs). Both heavy and light chains have three complementarity determining regions, called HCDR1, HCDR2, HCDR3 and LCDR1, LCDR2, and LCDR3 respectively.
  • the "antigen-binding fragment" of an antibody molecule refers to the amino acid fragment in the antibody molecule that participates in specific antigen binding, such as Fab, Fab' and (Fab') 2 , etc.
  • Single-chain antibodies and single-domain antibodies with antigen-binding ability are single-peptide chain antibody molecules and can be regarded as "antigen-binding fragments" of classic antibody molecules.
  • Single chain fragment variable is composed of an antibody heavy chain variable region and a light chain variable region connected through a short peptide to form a peptide chain. Through correct folding, the variable regions from the heavy chain and light chain form Fv segments through non-covalent interactions, so scFv can better retain its affinity activity for antigens.
  • Single domain antibody also known as “V HH antibody” refers to an antibody molecule with antigen-binding ability, including a heavy chain variable region but no light chain. From a structural point of view, single domain antibodies can also be considered as an antigen-binding fragment of an antibody molecule. It was first discovered in camelids. Subsequently, researchers discovered more single-domain antibodies with antigen-binding ability through screening of antibody libraries (such as phage display libraries). Single domain antibodies have some advantages over ordinary antibody molecules (for example, classic tetrameric antibody molecules) or their antigen-binding fragments, including but not limited to: smaller molecular weight, and when used in the human body, they can easily reach tissues that are difficult for ordinary antibody molecules to reach. or parts, or can access antigenic epitopes in proteins or polypeptides that are difficult for ordinary antibody molecules to access; they are more stable and can withstand changes in temperature and pH, as well as the effects of denaturants and proteases.
  • a “fully human antibody” refers to an antibody in which both the variable and constant regions (if any) are derived from human germline immunoglobulin sequences.
  • Fully human antibodies can be obtained through a variety of technologies, including phage antibody library technology, single B cell cloning technology, and transgenic mouse technology (for example, using the introduction of human germline immunoglobulin genes and removing the mouse's own germline immune globulin protein gene transgenic mice), etc.
  • animal-derived antibodies such as mouse-derived antibodies
  • fully human antibodies have the advantages of low immunogenicity and high safety when used in human patients.
  • Targeting or “specific binding” means that one molecule (e.g., an antibody or antigen-binding fragment thereof) has a higher affinity for another molecule (e.g., a tumor cell surface antigen) relative to other molecules also present in the environment. Binding affinity. "Targeting” or “specifically binding” does not exclude that the molecule may have binding affinity for more than one molecule, for example, a bispecific antibody may have high affinity for two different antigens.
  • GPRC5D is subtype D of the C5 family of G protein-coupled receptors. It is an orphan receptor and is a 7-transmembrane protein. Orphan receptors refer to receptors that are structurally similar to other confirmed receptors, but whose endogenous ligands have not yet been discovered. GPRC5D is highly expressed on the surface of primary multiple myeloma cells, while the expression in normal tissues is limited to the hair follicle area. Studies have shown that 65% of multiple myeloma patients have GPRC5D exceeding the 50% expression threshold. With this characteristic, GPRC5D has become potential target for the treatment of multiple myeloma (MM).
  • MM multiple myeloma
  • polypeptide and “protein” are used interchangeably and refer to a polymer of amino acid residues. Such polymers of amino acid residues may contain natural or unnatural amino acid residues and include, but are not limited to, peptides, oligopeptides, dimers, trimers and multimers of amino acid residues. Full-length proteins and their fragments are included in this definition. The term also includes post-expression modifications of the polypeptide, such as glycosylation, sialylation, acetylation, phosphorylation and similar modifications.
  • polypeptide refers to a protein that includes modifications to the native sequence, such as deletions, additions and substitutions (which are generally conservative in practice) so long as the protein retains the desired activity. These modifications may be purposeful, such as induced via site-directed mutagenesis, or may be accidental, such as via mutation of the host in which the protein is produced or errors due to PCR amplification.
  • nucleic acid molecule As used herein, the terms “nucleic acid molecule,” “nucleic acid,” and “polynucleotide” are used interchangeably to refer to polymers of nucleotides. Such nucleotide polymers may contain natural and/or non-natural nucleotides and include, but are not limited to, DNA, RNA and PNA. "Nucleic acid sequence” refers to a linear sequence of nucleotides contained in a nucleic acid molecule or polynucleotide.
  • vector refers to a nucleic acid molecule that can be engineered to contain a polynucleotide of interest (eg, a coding sequence for a polypeptide of interest) or a nucleic acid molecule that can replicate in a host cell (eg, nucleic acid, plasmid, or virus, etc.).
  • a vector may include one or more of the following components: an origin of replication, one or more regulatory sequences that regulate expression of a polynucleotide of interest (such as a promoter and/or enhancer), and/or one or more Selectable marker genes (such as antibiotic resistance genes and genes useful in colorimetric assays, such as beta-galactose).
  • expression vector refers to a vector used to express a polypeptide of interest in a host cell.
  • “Host cell” refers to a cell that is or has been the recipient of a vector or isolated polynucleotide.
  • the host cell can be a prokaryotic cell or a eukaryotic cell.
  • Exemplary eukaryotic cells include mammalian cells, such as primate or non-primate cells; fungal cells, such as yeast; plant cells; and insect cells.
  • Non-limiting exemplary mammalian cells include, but are not limited to, NSO cells, 293 and CHO cells, and derivatives thereof, such as 293-6E, CHO-DG44, CHO-K1, CHO-S and CHO-DS cells.
  • Host cells include the progeny of a single host cell, and the progeny may not necessarily be identical (in terms of morphology or genomic DNA complement) to the original parent cell due to natural, accidental, or deliberate mutations.
  • Host cells also include cells transfected in vivo with the nucleic acid molecules or expression vectors provided herein.
  • Treatment refers to the treatment of a subject to obtain a beneficial or desired clinical result.
  • Treatment encompasses a variety of treatments, including administration of any possible drug to the subject, surgery, radiation, etc.
  • beneficial or desired clinical outcomes include, but are not limited to, any one or more of the following: alleviation of one or more symptoms, attenuation of disease severity, prevention or delay of disease spread (e.g. metastasis, e.g. metastasize to the lungs or lymph nodes), prevent or delay disease recurrence, delay or slow down disease progression, improve disease conditions, inhibit disease or disease progression, block its development and remission (whether partial or complete remission).
  • the methods provided herein encompass any one or more of these aspects of treatment. In accordance with the above, “treatment” does not require complete removal of all symptoms of a condition or disease or complete alleviation.
  • terapéuticaally effective amount refers to an amount of active compound sufficient to elicit the biological or medical response desired by the clinician in a subject.
  • the "therapeutically effective dose” of the fusion protein of the present invention can be determined by those skilled in the art based on the route of administration, the subject's weight, age, condition and other factors. For example, a typical daily dosage may range from 0.01 mg to 100 mg or more of active ingredient per kg of body weight.
  • pharmaceutically acceptable carrier refers to solid or liquid diluents, fillers, antioxidants, stabilizers and the like that can be administered safely and are suitable for use by humans and/or Administration to the animal without undue adverse side effects while being suitable for maintaining the viability of the drug or active agent therein.
  • various carriers well known in the art may be administered, including, but not limited to, sugars, starch, cellulose and its derivatives, maltose, gelatin, talc, calcium sulfate, vegetable oils, synthetic oils, polyols , alginic acid, phosphate buffer, emulsifier, isotonic saline, and/or pyrogen-free water, etc.
  • the pharmaceutical compositions provided herein can be made into clinically acceptable dosage forms such as powders and injections.
  • composition of the present invention can be administered to a subject by any appropriate route, for example, by oral administration, intravenous infusion, intramuscular injection, subcutaneous injection, subperitoneal, rectal, sublingual, or by inhalation, transdermal, etc. route of administration.
  • Subject refers to an animal, such as a mammal, including (but not limited to) humans, rodents, simians, felines, canines, equines, bovines, porcines, sheep, goats, mammals Laboratory animals, mammalian farm animals, mammalian sporting animals and mammalian pets.
  • the subject may be male or female and may be of any appropriate age, including infants, juveniles, young adults, adults, and geriatric subjects.
  • a subject refers to an individual in need of treatment of a disease or condition.
  • a subject receiving treatment can be a patient who has a condition associated with the treatment, or is at risk of developing the condition.
  • the subject is a human, such as a human patient. The term is often used interchangeably with "patient,” "subject,” “subject,” etc.
  • sequence identity refers to the identity between two amino acid or nucleotide sequences (such as a query sequence and a reference sequence) A quantity of degree, usually expressed as a percentage. Usually, before calculating the percent identity between two amino acid or nucleotide sequences, the sequences are aligned and gaps (if any) are introduced. If at a certain alignment position, the amino acid residues or bases in the two sequences are the same, the two sequences are considered to be consistent or matching at that position; if the amino acid residues or bases in the two sequences are different, the two sequences are considered to be inconsistent or matching at that position. mismatch.
  • the number of matching positions is divided by the total number of positions in the alignment window to obtain sequence identity.
  • the number of gaps and/or the gap length is also taken into account.
  • the publicly available alignment software BLAST available on the web page ncbi.nlm.nih.gov
  • BLAST can be used to obtain an optimal sequence alignment and calculate two amino acids or nucleotides by using default settings Sequence identity between sequences.
  • Chimeric antigen receptor also known as chimeric T cell receptor, chimeric immune receptor
  • Chimeric antigen receptor is an engineered protein receptor molecule that can confer desired specificity to immune effector cells, such as with The ability to bind to specific tumor antigens.
  • Chimeric antigen receptors usually consist of an extracellular antigen-binding domain, a transmembrane domain, and an intracellular signaling domain. In most cases, the antigen-binding domain is a scFv sequence that is responsible for recognizing and binding to a specific antigen.
  • Intracellular signaling domains usually include immunoreceptor tyrosine activation motifs (ITAMs), such as the signaling domain derived from CD3 ⁇ molecules, which are responsible for activating immune effector cells and producing killing effects.
  • ITAMs immunoreceptor tyrosine activation motifs
  • the chimeric antigen receptor may also include a signal peptide at the amino terminus responsible for the intracellular localization of the nascent protein, and a hinge region between the antigen-binding domain and the transmembrane domain.
  • intracellular signaling domains may also include costimulatory domains derived, for example, from the 4-1BB molecule.
  • CAR-T cells refer to T cells expressing CAR, which are usually obtained by transducing T cells with an expression vector encoding CAR.
  • Commonly used expression vectors are viral vectors, such as lentiviral expression vectors.
  • Chimeric antigen receptor-modified T cells (CAR-T) are not restricted by the major histocompatibility complex and have specific targeting killing activity and the ability to sustain expansion.
  • expression vectors encoding CAR can also be used to transform other lymphocytes such as NK cells to obtain targeted killer cells expressing the CAR.
  • Self-cleaving peptides refer to short peptides that can achieve the function of cleaving proteins through ribosome skipping rather than proteolytic hydrolysis, and may include T2A, F2A, P2A, etc.
  • colony stimulating factor 2 receptor subunit alpha colony stimulating factor 2 receptor subunit alpha
  • EGFRt or tEGFR are used interchangeably herein to refer to a gene encoding a truncated human epidermal growth factor receptor polypeptide, or its encoded product, which lacks the distal membrane EGF binding domain and cytoplasmic signaling tail but retains Extracellular epitopes recognized by anti-EGFR antibodies.
  • EGFRt can be used as a non-immunogenic selection tool as well as a tracking marker with the function of genetically modifying cells.
  • the CAR-T cells provided in this article can also be considered for use in combination with safety switches of other mechanisms.
  • the CARs provided herein can be engineered so that they can utilize chemical inducers of dimerization (CIDs) to control CAR activity.
  • CIDs chemical inducers of dimerization
  • a complete CAR provided herein can be expressed as a separate fusion protein (e.g., the CD3 ⁇ intracellular domain is expressed separately from other parts) and utilizes a FKBP/FRB-based binding domain and rapamycin (The dimerization mechanism of rapamycin or rapamycin analogs) reconstitutes the intact CAR within the cell, allowing the integrity and activity of the CAR to be controlled by the presence or absence of rapamycin (e.g., see description in U.S. Patent 11,084,880) .
  • CAR-T cell activity can be controlled by degrading CAR molecules or causing CAR-T cell death. Examples of this include the use of PROTAC technology to degrade CAR molecules and the use of proteins with the ability to induce apoptosis, such as caspase-9 [8] .
  • SEQ ID NO: 1 is the amino acid sequence of the CDR1 cloned in #18, which is the CDR1 of the extracellular antigen-binding domain of GPRC5D CAR 2290.
  • SEQ ID NO: 2 is the amino acid sequence of the CDR2 of clone #18, which is the CDR2 of the extracellular antigen-binding domain of GPRC5D CAR 2290.
  • SEQ ID NO: 3 is the amino acid sequence of the CDR3 of clone #18, which is the CDR3 of the extracellular antigen-binding domain of GPRC5D CAR 2290.
  • SEQ ID NO: 4 is the amino acid sequence of the CDR1 cloned in #39, which is the CDR1 of the extracellular antigen-binding domain of GPRC5D CAR 2436.
  • SEQ ID NO: 5 is the amino acid sequence of the CDR2 cloned in #39, which is the CDR2 of the extracellular antigen-binding domain of GPRC5D CAR 2436.
  • SEQ ID NO: 6 is the amino acid sequence of the CDR3 cloned in #39, which is the CDR3 of the extracellular antigen-binding domain of GPRC5D CAR 2436.
  • SEQ ID NO: 7 is the amino acid sequence of the CDR1 cloned in #41, which is the CDR1 of the extracellular antigen-binding domain of GPRC5D CAR 2438.
  • SEQ ID NO: 8 is the amino acid sequence of the CDR2 cloned in #41, which is the CDR2 of the extracellular antigen-binding domain of GPRC5D CAR 2438.
  • SEQ ID NO: 9 is the amino acid sequence of the CDR3 cloned in #41, which is the CDR3 of the extracellular antigen-binding domain of GPRC5D CAR 2438.
  • SEQ ID NO: 10 is the amino acid sequence of the VHH of clone #18, that is, the sequence of the extracellular antigen-binding domain of GPRC5D CAR 2290.
  • SEQ ID NO: 11 is the amino acid sequence of VHH cloned in #39, that is, the sequence of the extracellular antigen-binding domain of GPRC5D CAR 2436.
  • SEQ ID NO: 12 is the amino acid sequence of the VHH of clone #41, that is, the sequence of the extracellular antigen-binding domain of GPRC5D CAR 2438.
  • SEQ ID NO: 13 is the coding sequence of the VHH of clone #18, that is, the coding sequence of the extracellular antigen-binding domain of GPRC5D CAR 2290.
  • SEQ ID NO: 14 is the coding sequence of VHH cloned in #39, that is, the coding sequence of the extracellular antigen-binding domain of GPRC5D CAR 2436.
  • SEQ ID NO: 15 is the coding sequence of the VHH of clone #41, that is, the coding sequence of the extracellular antigen-binding domain of GPRC5D CAR 2438.
  • SEQ ID NO: 16 is the nucleic acid sequence encoding the CD8 ⁇ signal peptide.
  • the CD8 ⁇ signal peptide can be used to guide newly synthesized CAR to the cell surface. In mature CAR, the signal peptide can be cleaved, so the signal peptide may not be included.
  • SEQ ID NO: 17 is the amino acid sequence of CD8 ⁇ signal peptide.
  • SEQ ID NO: 18 is the nucleic acid sequence encoding the CD8 ⁇ hinge region.
  • the CD8 ⁇ hinge region can be used to connect the extracellular antigen-binding domain and the transmembrane domain in the CAR.
  • SEQ ID NO: 19 is the amino acid sequence of the CD8 ⁇ hinge region.
  • SEQ ID NO: 20 is the nucleic acid sequence encoding the CD8 ⁇ transmembrane region.
  • SEQ ID NO: 21 is the amino acid sequence of the CD8 ⁇ transmembrane region.
  • SEQ ID NO: 22 is the nucleic acid sequence encoding the 4-1BB intracellular domain.
  • SEQ ID NO: 23 is the amino acid sequence of the 4-1BB intracellular domain.
  • SEQ ID NO: 24 is the nucleic acid sequence encoding the intracellular signaling domain of CD3 ⁇ .
  • SEQ ID NO: 25 is the amino acid sequence of the CD3 ⁇ intracellular signaling domain.
  • SEQ ID NO: 26 is the nucleic acid sequence encoding the self-cleaving peptide T2A.
  • SEQ ID NO: 27 is the amino acid sequence of self-cleaving peptide T2A. Due to the existence of self-cleaving peptide, the proteins on both sides of the self-cleaving peptide (CAR and EGFRt) are separated from each other during the translation process. For convenience of description, it can be considered that a CAR including multiple elements as a fusion protein may also include T2A and EGFRt.
  • SEQ ID NO: 28 is the nucleic acid sequence encoding the CSF2RA signal peptide.
  • SEQ ID NO: 29 is the amino acid sequence of the CSF2RA signal peptide. Can be located upstream of EGFRt to guide newly synthesized EGFRt to the cell surface. The CSF2RA signal peptide can be cleaved away from EGFRt.
  • SEQ ID NO: 30 is the coding nucleic acid sequence of EGFRt.
  • SEQ ID NO: 31 is the amino acid sequence of EGFRt.
  • SEQ ID NO: 32 is the nucleic acid sequence encoding the self-cleaving peptide P2A.
  • the self-cleaving peptide P2A can also be selected to replace T2A as the connecting peptide between CAR and EGFRt.
  • SEQ ID NO: 33 is the amino acid sequence of self-cleaving peptide P2A.
  • SEQ ID NO: 34 is the coding sequence of GPRC5D CAR 2290 (#18).
  • SEQ ID NO: 35 is the amino acid sequence of GPRC5D CAR 2290 (#18).
  • SEQ ID NO: 36 is the coding sequence of GPRC5D CAR 2436 (#39).
  • SEQ ID NO: 37 is the amino acid sequence of GPRC5D CAR 2436 (#39).
  • SEQ ID NO: 38 is the coding sequence of GPRC5D CAR 2438 (#41).
  • SEQ ID NO: 39 is the amino acid sequence of GPRC5D CAR 2438 (#41).
  • sequences shown in SEQ ID NO: 34-39 are only the coding or amino acid sequence of the CAR structure and do not contain the EGFRt element.
  • clones #18, #39 and #41 are examples of specific single domain antibodies targeting GPRC5D provided in this application for CAR construction.
  • the clones used for CAR construction also include single domain antibodies #14 and #10. , and #8, as well as scFv#46, #47, etc.
  • the chimeric antigen receptor (CAR) provided herein is an artificially constructed chimeric protein, including an extracellular antigen-binding domain that specifically binds GPRC5D, a transmembrane domain, and one or more intracellular signaling structures. area. Characteristics of these CARs include their ability to redirect T cell specificity and responsiveness to GPRC5D-expressing cells in a non-MHC-restricted manner. Non-MHC restricted GPRC5D recognition enables T cells or NK cells expressing the disclosed CAR to recognize antigen independent of antigen processing.
  • Intracellular signaling domains may include T cell receptor signaling domains, T cell costimulatory signaling domains, or both.
  • the T cell receptor signaling domain may include the intracellular domain of the T cell receptor, such as the intracellular portion of the CD3 ⁇ protein.
  • Costimulatory signaling domains are intracellular domains of costimulatory molecules. Costimulatory molecules are cell surface molecules other than the antigen receptor or its ligand that are required for effective lymphocyte response to antigen.
  • a CAR provided herein includes an extracellular antigen binding domain that specifically binds GPRC5D.
  • the antigen-binding domain may be a scFv that specifically binds to the heavy and light chain variable regions of any antibody or antigen-binding fragment thereof linked by a peptide linker to GPRC5D.
  • the extracellular antigen-binding domain may include the V HH from the single domain antibody without including any light chain variable region.
  • the extracellular antigen binding domain of the CAR provided herein includes a V HH from clone #18, the sequences of HCDR1, HCDR2 and HCDR3 of the above #18 V HH are as follows:
  • HCDR1 The sequence of HCDR1 is GGSFSGYY (SEQ ID NO: 1);
  • HCDR2 The sequence of HCDR2 is INHSGST (SEQ ID NO: 2);
  • HCDR3 The sequence of HCDR3 is ARARRYGGRTRFDP (SEQ ID NO: 3).
  • the extracellular antigen binding domain of the CAR provided herein includes a V HH from clone #39, the sequences of HCDR1, HCDR2 and HCDR3 of the above #39 V HH are as follows:
  • HCDR1 The sequence of HCDR1 is GFIFSSYG (SEQ ID NO: 4);
  • HCDR2 The sequence of HCDR2 is ISSSGDYT (SEQ ID NO: 5);
  • HCDR3 The sequence of HCDR3 is ARMSFRRYDH (SEQ ID NO: 6).
  • the extracellular antigen binding domain of the CAR provided herein includes a V HH from clone #18, and the sequences of HCDR1, HCDR2 and HCDR3 of the above #41 V HH are as follows:
  • HCDR1 The sequence of HCDR1 is GFSFSGYI (SEQ ID NO: 7);
  • HCDR2 The sequence of HCDR2 is TSSSGTET (SEQ ID NO: 8);
  • HCDR3 is ARYYSKYGRSYHVDS (SEQ ID NO: 9).
  • the extracellular antigen binding domain of a CAR provided herein includes VHH (SEQ ID NO: 10) from clone #18, or any functional variant thereof.
  • the CAR extracellular antigen binding domain provided herein includes VHH from clone #39 (SEQ ID NO: 11) or any functional variant thereof.
  • the CAR extracellular antigen binding domain provided herein includes VHH from clone #41 (SEQ ID NO: 12) or any functional variant thereof.
  • Functional variants herein may include the insertion, deletion or substitution of several amino acid residues compared to their parent sequence (the VH amino acid sequence provided herein) and substantially retain the ability to specifically bind GPRC5D.
  • functional variants may include 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16 as compared to their parent sequence , 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30 or more amino acid residues, while retaining the identity of the parent sequence and GPRC5D
  • the binding affinity is at least 50%, 60%, 70%, 80%, 90%, or even higher than the parent sequence.
  • the VH framework sequence can have considerable room for change, as long as it can maintain the spatial position of the corresponding CDR sequence.
  • the framework region sequence in the VH amino acid sequence can be changed, for example, by replacing the framework regions of antibody molecules of different species with each other, and functional variants that substantially retain the antigen-binding ability can be obtained.
  • the extracellular antigen-binding domain of a CAR may include two or more VHH fragments described above with GPRC5D binding ability. They can be connected in series directly or via a peptide linker. Using two or more VHH fragments connected in series as the extracellular antigen-binding domain or part thereof helps to promote the recognition and binding of the constructed CAR to target molecules (such as GPRC5D). Preferably, these VH fragments each bind to different epitopes on GPRC5D.
  • the CAR may include a signal peptide sequence, for example, located N-terminal to the antigen-binding domain. Any suitable signal peptide sequence can be used.
  • the signal peptide sequence is a CD8 ⁇ signal peptide.
  • the signal peptide sequence can promote the expression of CAR on the cell surface, the presence of the signal peptide sequence in the expressed CAR is not necessary for the CAR to function.
  • the signal peptide sequence may be cleaved from the CAR.
  • a CAR lacks a signal peptide sequence.
  • a hinge region (or spacer domain), which includes the polypeptide sequence.
  • the hinge region may contain up to 300 amino acids, preferably 10 to 100 amino acids.
  • a CAR provided herein includes a CD8 alpha protein hinge region.
  • the CAR may include a transmembrane domain fused to the extracellular domain of the CAR.
  • transmembrane domains that are naturally associated with other elements in the CAR, such as cross-linking regions or intracellular signaling domains, are used.
  • Transmembrane domains can be derived from natural or synthetic sources. Where the source is natural, the domain may be derived from any membrane-binding or transmembrane protein. Exemplary transmembrane domains for use in the disclosed CARs may include at least the alpha, beta or zeta chain of a T cell receptor, CD28, CD3 ⁇ , CD45, CD4, CD5, CDS, CD9, CD 16, CD22, CD33, Transmembrane domains of CD37, CD64, CD80, CD86, CD 134, CD137, and CD154. Alternatively, the transmembrane domain may be synthetic, in which case it will contain primarily hydrophobic residues such as leucine and valine. In some embodiments, a triplet of phenylalanine, tryptophan, and valine will be present at each end of the synthetic transmembrane domain.
  • a short oligopeptide or polypeptide linker may form the link between the transmembrane domain and the intracellular T cell signaling domain and/or T cell costimulatory domain of the CAR.
  • exemplary linker sequences include one or more glycine-serine doublets.
  • the transmembrane domain includes a transmembrane domain of a T cell receptor, such as the CD8 alpha transmembrane domain (SEQ ID NO: 21).
  • the intracellular region of the CAR includes one or more intracellular T cell signaling domains responsible for activating at least one normal effector function of the CAR-expressing T cells.
  • Exemplary T cell signaling domains are provided herein and are known to those of ordinary skill in the art.
  • the entire intracellular T cell signaling domain can be used in a CAR, in many cases it is not necessary to use the entire chain. To the extent that a truncated portion of an intracellular T cell signaling domain is used, the truncated portion can be used in place of the intact chain as long as it transduces the relevant T cell effector function signal.
  • intracellular T cell signaling domains used in CARs include the cytoplasmic sequences of the T cell receptor (TCR) and costimulatory molecules that cooperate to initiate signal transduction upon antigen receptor binding, as well as any of these sequences Derivatives or variants and any synthetic sequence having the same function.
  • TCR T cell receptor
  • T cell receptor signaling domains regulate activation of T cell receptor complexes in a stimulatory or inhibitory manner.
  • the CARs disclosed herein may include cytoplasmic signaling sequences that act in a stimulatory manner, which may contain signaling motifs known as immunoreceptor tyrosine-based activation motifs or ITAMs.
  • ITAM-containing primary cytoplasmic signaling sequences that may be included in the disclosed CARs include intracellular domains from CD3 ⁇ , FcR ⁇ , FcR ⁇ , CD3 ⁇ , CD3 ⁇ , CD3 ⁇ , CDS, CD22, CD79a, CD79b, and CD66d proteins.
  • the cytoplasmic signaling molecule in the CAR includes the intracellular T cell signaling domain from CD3 ⁇ .
  • the intracellular domain of the CAR provided herein may include a CD3 zeta chain portion and an intracellular costimulatory signaling domain.
  • the costimulatory signaling domain may include an intracellular domain of a costimulatory molecule.
  • Costimulatory molecules are cell surface molecules other than the antigen receptor or its ligand that are required for effective lymphocyte response to antigen. Examples of such molecules include CD27, CD28, 4-1BB (CD137), OX40 (CD134), CD30, CD40, PD-1, ICOS, Lymphocyte Function Associated Antigen 1 (LFA-1), CD2, CD7, LIGHT, NKG2C and B7-H3. .
  • a CAR can include a CD3 ⁇ signaling domain, a CD8 signaling domain, a CD28 signaling domain, a 4-1BB signaling domain, or a combination of two or more thereof.
  • the intracellular domain includes the signaling domain of CD3- ⁇ and the signaling domain of CD28.
  • the cytoplasmic domain includes the signaling domain of CD3 ⁇ and the signaling domain of 4-1BB.
  • the cytoplasmic domain includes the signaling domain of CD3- ⁇ and the signaling domains of CD28 and CD137.
  • the cytoplasmic signaling sequences within the cytoplasmic signaling portion of the CAR of the invention can be linked to each other in random or specified order.
  • short polypeptide linkers preferably between 2 and 10 amino acids in length, can form the linkage. Glycine-serine doublets provide particularly suitable linkers.
  • a spacer domain which includes the polypeptide sequence.
  • the spacer domain may comprise up to 300 amino acids, preferably 10 to 100 amino acids, and most preferably 25 to 50 amino acids.
  • CAR a parent CAR
  • those skilled in the art can modify it, for example, fuse it with other polypeptides, or retain only fragments of the CAR provided herein, and these fusion molecules or fragments have the parent CAR
  • At least part of the biological activity of the CAR such as the activity of recognizing target cells (eg, tumor cells expressing GPRC5D) or detecting, treating, or preventing disease.
  • the CARs provided herein may also include additional amino acids at the amino or carboxyl terminus, or at both termini, that are not present in the amino acid sequence of the parent CAR.
  • the additional amino acids do not interfere with the biological function of the CAR or functional portion, such as identifying target cells, detecting cancer, treating or preventing cancer, etc. More ideally, the additional amino acid enhances the biological activity compared to the biological activity of the parent CAR.
  • Functional variants of the CARs described herein that have substantial or significant sequence identity or similarity to the parent CAR, which functional variants retain the biological activity of the CAR of which they are a variant.
  • Functional variants encompass, for example, those variants of the CARs described herein (parental CAR) that retain the ability to recognize target cells to a similar degree, to the same degree, or to a greater degree than the parental CAR.
  • the functional variant may be, for example, at least about 30%, about 50%, about 75%, about 80%, about 85%, about 90%, about 91%, about 92% identical in amino acid sequence to the parent CAR. , about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, about 99% or higher consistency.
  • Functional variants may, for example, include the amino acid sequence of a parent CAR with at least one conservative amino acid substitution.
  • functional variants may include the amino acid sequence of the parent CAR with at least one non-conservative amino acid substitution. In this case, it is preferred that the non-conservative amino acid substitutions do not interfere with or inhibit the biological activity of the functional variant. Non-conservative amino acid substitutions can enhance the biological activity of the functional variant, making the biological activity of the functional variant increased compared with the parental CAR.
  • CARs provided herein may include synthetic amino acids in place of one or more naturally occurring amino acids.
  • Such synthetic amino acids are known in the art and include, for example, aminocyclohexanecarboxylic acid, norleucine, a-amino-n-decanoic acid, homoserine, S-acetamidomethyl-cysteine, trans- 3- and trans-4-hydroxyproline, 4-aminophenylalanine, 4-nitrophenylalanine, 4-chlorophenylalanine, 4-carboxyphenylalanine, ⁇ -phenyl Serine, ⁇ -hydroxyphenylalanine, phenylglycine, ⁇ -naphthylalanine, cyclohexylalanine, cyclohexylglycine, indoline-2-carboxylic acid, 1,2,3,4-tetrakis Hydroisoquinoline-3-carboxylic acid, aminomalonic acid, aminomalonic acid monoamide, N'-benzyl
  • CARs provided herein can be glycosylated, amidated, carboxylated, phosphorylated, esterified, N-acylated, cyclized via, for example, disulfide bonds, or converted to acid addition salts and/or optionally dimerized Either polymerization or conjugation.
  • nucleic acid molecules encoding the disclosed chimeric antigen-binding receptors can be included in expression vectors (eg, lentiviral vectors) for expression in host cells, such as T cells, to prepare the disclosed CARs.
  • methods of using chimeric antigen receptors include isolating T cells from a subject, transforming the T cells with an expression vector encoding the chimeric antigen receptor (eg, a lentiviral vector), and expressing the chimeric antigen The recipient's engineered T cells are administered to the subject for treatment, for example, to treat a tumor in the subject.
  • nucleic acid sequences encoding various elements for constructing a CAR.
  • These nucleic acid sequences may be prepared by any suitable method, including, for example, cloning, amplification, or direct chemical synthesis. Chemical synthesis produces single-stranded oligonucleotides. This can be converted to double-stranded DNA by hybridization to complementary sequences or by polymerization with DNA polymerase using the single strand as a template. The skilled person will recognize that although chemical synthesis of DNA is generally limited to sequences of about 100 bases, longer sequences can be obtained by joining shorter sequences.
  • Exemplary nucleic acids can be prepared through cloning techniques. Examples of appropriate cloning and sequencing techniques are known, as well as instructions sufficient to guide the technician through many cloning exercises. Nucleic acids can also be prepared by amplification methods. Amplification methods include polymerase chain reaction (PCR), ligase chain reaction (LCR), transcription-based amplification system (TAS), and self-sustaining sequence replication system (3SR). A variety of cloning methods, host cells, and in vitro amplification methods are well known to those skilled in the art.
  • PCR polymerase chain reaction
  • LCR ligase chain reaction
  • TAS transcription-based amplification system
  • 3SR self-sustaining sequence replication system
  • a nucleic acid molecule encodes a CAR provided herein for expression in a T cell to generate a chimeric antigen receptor T cell.
  • Nucleic acid molecules encoding chimeric antigen-binding receptors can be contained in vectors (eg, lentiviral vectors) for expression in host cells, such as T cells.
  • Exemplary cells include T cells, natural killer (NK) cells, cytotoxic T lymphocytes (CTL), and regulatory T cells.
  • Nucleic acid molecules can be expressed in recombinantly engineered cells such as bacterial, plant, yeast, insect, and mammalian cells.
  • Antibodies, antigen-binding fragments can be expressed as VHH alone (for single domain antibodies), or VH and VL chains or can be expressed as fusion proteins.
  • the DNA fragment encoding VH and VL can be operably linked to another fragment encoding a flexible linker, e.g., encoding the amino acid sequence (Gly 4 -Ser) 3 , such that the VH and VL sequences can be expressed It is a continuous single-chain protein whose V L and V H domains are connected by this flexible linker.
  • a flexible linker e.g., encoding the amino acid sequence (Gly 4 -Ser) 3 , such that the VH and VL sequences can be expressed It is a continuous single-chain protein whose V L and V H domains are connected by this flexible linker.
  • E. coli E. coli
  • other bacterial hosts E. coli
  • yeast E. coli
  • various higher eukaryotic cells such as COS, CHO, HeLa, and myeloma cell lines.
  • host cells are preferably mammalian cells, such as human cells.
  • the host cells may be peripheral blood lymphocytes (PBL) or peripheral blood mononuclear cells (PBMC), or T cells.
  • the T cell can be any T cell, such as a cultured T cell, such as a primary T cell, or a T cell from a cultured T cell line, such as Jurkat, SupTl, etc., or a T cell obtained from a mammal. If obtained from a mammal, T cells can be obtained from a variety of sources, including but not limited to blood, bone marrow, lymph nodes, thymus, or other tissues or body fluids. T cells can also be enriched or purified.
  • the T cells can be human T cells.
  • the T cells may be T cells isolated from humans.
  • T cells can be any type of T cell and can be at any stage of development, including but not limited to CD4 + /CD8 + double positive T cells, CD4 + helper T cells such as Th 1 and Th 2 cells, CD8 + T cells (e.g. , cytotoxic T cells), tumor infiltrating cells, memory T cells, naive T cells, etc.
  • T cells can be CD8 + T cells or CD4 + T cells.
  • Nucleic acids encoding a CAR or elements thereof described herein can be modified without reducing their biological activity. For example, some modifications can be made to facilitate cloning or expression. Such modifications are well known to those skilled in the art and include, for example, a stop codon, a methionine added at the amino terminus to provide a starting site, additional amino acids at either terminus to create conveniently positioned restriction sites (such as polyhistidine) to aid in the purification step.
  • the CARs of the present disclosure, or elements thereof may also be constructed in whole or in part using standard peptide synthesis well known in the art.
  • the CARs or CAR-expressing host cells (such as T cells or NK cells) provided herein can specifically recognize and bind to target cells expressing GPRC5D, such as tumor cells.
  • compositions for preventing or treating diseases associated with GPRC5D expression which include host cells, particularly CAR-T cells, expressing the CAR provided herein, and pharmaceutically acceptable Carrier.
  • host cells particularly CAR-T cells, expressing the CAR provided herein, and pharmaceutically acceptable Carrier.
  • These pharmaceutical compositions can be formulated into dosage forms suitable for administration, such as injection solutions.
  • kits for treating diseases associated with GPRC5D expression comprising administering an effective amount of a CAR-T cell or a pharmaceutical composition comprising a CAR-T cell provided herein to a subject in need thereof.
  • the administration method is intravenous administration, such as intravenous injection; intramuscular administration, such as intramuscular injection; or in situ administration to the tumor part, etc.
  • provided herein are uses of the above-mentioned CAR-T cells in the preparation of medicaments for treating diseases associated with GPRC5D expression.
  • the disease associated with GPRC5D expression is a neoplasm, such as a plasma cell malignant disease (e.g., multiple myeloma) or a B cell malignant disease (e.g., Hodgkin lymphoma or non-Hodgkin lymphoma); or autoimmune disease.
  • a plasma cell malignant disease e.g., multiple myeloma
  • a B cell malignant disease e.g., Hodgkin lymphoma or non-Hodgkin lymphoma
  • autoimmune disease e.g., a neoplasm, such as a plasma cell malignant disease (e.g., multiple myeloma) or a B cell malignant disease (e.g., Hodgkin lymphoma or non-Hodgkin lymphoma); or autoimmune disease.
  • the CAR-T cells or pharmaceutical compositions herein can be administered to a subject to prevent or treat tumors.
  • a therapeutically effective amount of CAR-T cells or pharmaceutical compositions targeting GPRC5D-expressing target cells is administered to a subject, thereby slowing or inhibiting tumor growth or metastasis, reducing tumor volume, or inhibiting cancer. signs or symptoms.
  • the therapeutically effective amount will depend on the severity of the disease and the general health of the patient.
  • a therapeutically effective amount is an amount that provides subjective relief of symptoms or an objectively identifiable improvement as noted by a clinician or other qualified observer.
  • a therapeutically effective amount is an amount necessary to inhibit tumor growth, inhibit metastasis, reduce tumor volume, or is effective to reduce signs or symptoms of a tumor.
  • the therapeutically effective amount of an agent administered can vary depending on the desired effect and the subject being treated.
  • a therapeutic amount is an amount that eliminates or reduces tumor burden in a patient, or prevents or reduces metastatic cell proliferation, or alleviates tumor symptoms.
  • any method of administration may be used for the disclosed CAR-T cells or pharmaceutical compositions, including local and systemic administration.
  • topical, oral, intravascular (eg, intravenous), intramuscular, intraperitoneal, intranasal, intradermal, intrathecal and subcutaneous administration may be used.
  • intravascular eg., intravenous
  • intramuscular e.g., intraperitoneal
  • intranasal e.g., intradermal, intrathecal
  • subcutaneous administration e.g., topical, oral, intravascular (eg, intravenous), intramuscular, intraperitoneal, intranasal, intradermal, intrathecal and subcutaneous administration may be used.
  • the specific mode of administration and dosing regimen will be selected by the attending clinician, taking into account the specific circumstances of the case (e.g., subject, disease, disease state involved, and whether treatment is prophylactic).
  • Administration of CAR-T cells or pharmaceutical compositions provided herein may be concomitant with the administration of other anti-cancer or anti-angiogenic agents or therapeutic treatments (eg, surgical resection of tumors or radiation therapy).
  • the subject can receive one or more additional therapies or therapeutic agents before or after administration of a therapeutically effective amount of CAR-T cells.
  • the additional therapy may include, but is not limited to, the use of chemotherapeutic agents, anti-angiogenic agents, or combinations thereof.
  • at least a portion of the tumor is removed or its size or volume is reduced by surgery or other means prior to administration of an effective amount of a CAR-T cell or pharmaceutical composition provided herein.
  • additional therapeutic agents include microtubule binding agents, DNA intercalating or cross-linking agents, DNA synthesis inhibitors, DNA and RNA transcription inhibitors, antibodies, enzymes, enzyme inhibitors, gene modulators, and angiogenesis Inhibitors. These agents (administered in therapeutically effective amounts) and treatments may be used alone or in combination.
  • any suitable anti-cancer agent or anti-angiogenic agent can be administered in combination with the CAR-T cells or pharmaceutical compositions disclosed herein.
  • the present invention uses a fully human phage library to screen antibodies and directly obtain fully human monoclonal antibodies. Compared with traditional hybridoma technology, it eliminates the difficult step of humanizing mouse antibodies, and fully human antibodies have lower immunogenicity than humanized mouse antibodies. In the development of antibody drugs or CAR-T There is potential for better.
  • Antibody clones that specifically bind to the cell surface GPRC5D antigen were obtained through a fully human phage library (including single domain antibodies #18, #39, #41, #14, #10, #8 and scFv #46, #47, etc.). These clones and the control antibody benchmark 1 (the variable region sequence comes from GC5B596 of Chinese Patent Application Publication CN 109715667 A) were then constructed on the second-generation CAR structure, and then lentivirus packaging was carried out and transfected into T cells. In the CAR-T At the cellular level, fully human GPRC5D antibody clones and candidate CAR-T molecules with stronger functions than the control benchmark1 were screened from the perspectives of target cell activation and killing, and target cell stimulation and proliferation.
  • V HH heavy chain variable region sequences
  • CD107a degranulation assay CD107a degranulation assay
  • in vitro cell killing assay in vitro cytotoxicity assay
  • T cells were transfected with the CAR lentivirus with the structure shown in Figure 1 and EGFRt expression was detected 5 to 7 days later ( Figure 2).
  • the cells were divided into two batches for transfection.
  • the CAR structure contains CD8 ⁇ signal peptide, VHH or scFv, CD8 ⁇ hinge region, CD8 ⁇ transmembrane region, 4-1BB co-stimulatory molecule and CD3 ⁇ , and uses T2A to connect a truncated EFGR molecule (EGFRt), which can be used as a safety agent in clinical translation. switch is used. Since EGFRt is co-expressed with CAR molecules, it can be used as an indirect detection indicator of the distribution of CAR molecules on the surface of T cells without affecting the structure and function of CAR.
  • EGFRt truncated EFGR molecule
  • CD107a is a marker of intracellular microvesicles. When granzyme-loaded microvesicles fuse with the cell membrane, CD107a on the cell membrane will increase. When its recycling is blocked with monesin (purchased from BioLegend) , which can quantitatively reflect the intensity of microvesicle release. When CAR-T is stimulated by the target antigen on the target cell, it will cause the release of granzymes, and the activation of T cells can be judged by flow cytometric detection of the increase in CD107a.
  • monesin purchased from BioLegend
  • CAR-T cells were obtained through lentiviral transduction, and the CAR-T cells were cultured in vitro for 9-12 days before performing a CD107a degranulation experiment.
  • the CAR-T cells to be tested were incubated with target cells, monensin and CD107a antibodies for 4 hours.
  • the cell densities of CAR-T cells and target cells were both 2 ⁇ 10 5 cells/mL.
  • the samples were labeled with CD8 antibodies and EGFR antibodies, and flow cytometric detection was performed.
  • the degranulation levels of GPRC5D-positive cells in clone numbers 18, 39, and 41 were higher, and all were higher than Benchmark 1.
  • the degranulation level of the same clone numbers 18 and 46 and 47 in CAR cells was also higher than that of Benchmark 1.
  • the degranulation level of clone number 14 was slightly weaker.
  • the degranulation level of the same clone numbers 18 and 10 in CAR cells was also higher than that of Benchmark 1.
  • Table 1 Results of CD107a degranulation by different target cells on CAR-T cells in each group.
  • U266 MM1.s 8226 was used as the positive target cell of GPRC5D, and Raji and CCRF cells were used as negative target cells of GPRC5D to evaluate the antigen-specific killing ability of GPRC5D CAR-T cells.
  • the above target cells were each obtained through lentiviral transduction to obtain target cells stably expressing firefly luciferase. Therefore, the luciferase activity in the sample can reflect the number of target cells.
  • CAR-T cells and target cells are co-incubated and cultured.
  • luciferase When target cells are killed by CAR-T cells, luciferase will be released and quickly inactivated (the half-life of firefly luciferase is about 0.5 hours). If the target cells are not killed or inhibited by CAR-T cells, more luciferase will be produced as the target cells expand and luciferase continues to be expressed. Therefore, the killing of target cells by CAR-T can be detected through luciferase activity.
  • the CAR-T cell sample and a fixed number of target cells (1x10 4 cells) were mixed according to different effective-to-target ratios (E:T) and incubated together for 24 hours, and then the luciferase activity (RLU) in the sample was detected. Since luciferase activity can reflect the number of target cells in the sample, the killing/inhibitory ability of CAR-T cells on target cells can be obtained through changes in luciferase activity in the sample. The lower the luciferase activity reading (RLU), the more target cells are killed.
  • E:T effective-to-target ratios
  • RLU luciferase activity
  • U266 MM1.s 8226 was used as the positive target cell of GPRC5D, and Raji and CCRF cells were used as negative target cells of GPRC5D.
  • GPRC5D CAR-T cells were incubated with the target cells, and the intracellular expression of IFN- ⁇ and TNF ⁇ in the CAR cells was determined by flow cytometry. .
  • TNF ⁇ and IFN- ⁇ were significantly increased after GPRC5D CAR-T was only co-incubated with GPRC5D-positive cells, but there was no obvious increase in TNF ⁇ and IFN- ⁇ after co-incubation with negative cell lines. expression levels increased. Among them, the expression levels of TNF ⁇ and IFN- ⁇ in GPRC5D-positive cells with clone numbers 18, 39, and 41 were higher, and both were higher than Benchmark 1. At the same time, the expression of TNF ⁇ and IFN- ⁇ on various positive target cells of these three clones is relatively uniform, and Benchmark 1 has lower secretion of these two cytokines under the stimulation of U266 and H929.
  • Mitomycin-treated target cells 8226 were mixed with different groups of GPRC5D CAR-T cells. After multiple stimulations, the CAR-T cells and target cells were co-incubated and cultured to determine the effects of different CAR-T cells on the target cells. The ability of target cells to proliferate after repeated stimulation.
  • Target cells 8226;
  • CAR-T cell samples can effectively expand after repeated stimulation. After 7 times of target cell stimulation, the expansion ability of clone numbers 18, 39, and 41 CAR-T cells is stronger than that of Benchmark1. .
  • the proliferation ability of CAR-T cells after being stimulated by target cells is closely related to the long-term prognosis of patients. Therefore, CAR clones 2290 (#18), 2436 (#39), and 2438 (#41) are considered to have stronger long-term proliferation and elimination potential of tumor cells.
  • Example 6 Determination of the performance of organ-derived tumor cells in stimulating CAR cell degranulation
  • CD107a is a marker of intracellular microvesicles. When granzyme-loaded microvesicles fuse with the cell membrane, CD107a on the cell membrane will increase. When its recycling is blocked with monesin (purchased from BioLegend) , which can quantitatively reflect the intensity of microvesicle release. When CAR-T is stimulated by the target antigen on the target cell, it will cause the release of granzyme, and the activation of T cells by the target cell can be judged by flow cytometry to detect the increase in CD107a.
  • monesin purchased from BioLegend
  • CAR-T cells were obtained through lentiviral transduction, and the CAR-T cells were cultured in vitro for 9-12 days before performing a CD107a degranulation experiment.
  • the CAR-T cells to be tested were incubated with target cells, monensin, and CD107a antibodies for 4 hours.
  • the cell densities of both CAR-T cells and target cells were 2 ⁇ 10 6 cells/mL.
  • the samples were labeled with CD8 antibodies and EGFR antibodies, and flow cytometric detection was performed.

Landscapes

  • Health & Medical Sciences (AREA)
  • Chemical & Material Sciences (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Organic Chemistry (AREA)
  • Medicinal Chemistry (AREA)
  • Immunology (AREA)
  • General Health & Medical Sciences (AREA)
  • Proteomics, Peptides & Aminoacids (AREA)
  • Molecular Biology (AREA)
  • Biophysics (AREA)
  • Biochemistry (AREA)
  • Genetics & Genomics (AREA)
  • Public Health (AREA)
  • Pharmacology & Pharmacy (AREA)
  • Veterinary Medicine (AREA)
  • Animal Behavior & Ethology (AREA)
  • Epidemiology (AREA)
  • Gastroenterology & Hepatology (AREA)
  • Microbiology (AREA)
  • Mycology (AREA)
  • Engineering & Computer Science (AREA)
  • Zoology (AREA)
  • Cell Biology (AREA)
  • Bioinformatics & Cheminformatics (AREA)
  • Toxicology (AREA)
  • Chemical Kinetics & Catalysis (AREA)
  • General Chemical & Material Sciences (AREA)
  • Nuclear Medicine, Radiotherapy & Molecular Imaging (AREA)
  • Micro-Organisms Or Cultivation Processes Thereof (AREA)
  • Peptides Or Proteins (AREA)

Abstract

本文提供了靶向GPRC5D的全人源嵌合抗原受体(CAR)。本文还提供了表达该CAR的宿主细胞，如CAR-T细胞。本文还提供了这些CAR和CAR-T细胞在肿瘤(例如多发性骨髓瘤)治疗方面的应用。

Description

靶向GPRC5D的全人源嵌合抗原受体(CAR)及其应用 技术领域
本申请涉及生物医药领域，具体涉及靶向GPRC5D的CAR和表达靶向GPRC5D的CAR的宿主细胞，以及它们的制备方法和应用。
背景技术
多发性骨髓瘤(multiple myleloma)是一种发生于骨髓的浆细胞肿瘤。该肿瘤会引发高钙血症，贫血，肾功能障碍，骨坏死和骨髓衰竭等病症。目前多发性骨髓瘤是第二大常见血液肿瘤 [1]。以2020年世界卫生组织WHO公布的多发性骨髓瘤发病率统计显示，亚洲人群发病率和死亡率占比分别为36％和42％，居所有洲统计的首位。全球多发性骨髓瘤的发病率和死亡率比为1.8:1.1，亚洲为1.1:0.76，可见多发性骨髓在发病人群的生存率较低。主要原因一是发生人群的年龄中位值偏高在66岁左右，40岁以下人群的发生率在2％左右。二是多发性骨髓瘤在常用的化疗，免疫调节剂以及单抗疗法下几乎无治愈可能性，勉强缓解也伴随着极高的复发率，即复发难治性骨髓瘤(Relapsed/refractory Multiple myeloma(RRMM)，五年生存率仅有51％ [1],[2]。
近年来在多发性骨髓瘤治疗上，T细胞双特异性抗体疗法bispecific T cell engagers(BiTEs)，和继发性T细胞疗法adoptive T cell therapy(ACT)取得了突破性进展。针对多发性骨髓瘤的靶向疗法产品靶点主要有BCMA、CD38、CD138、GPRC5D等 [2]。CD38和CD138会在正常组织的细胞上及造血干细胞上表达，利用靶向疗法清除后副作用较大，往往会对正常器官造成损伤或是自身免疫系统受损，而BCMA和GPRC5D主要表达在浆细胞或骨髓瘤中的浆细胞上，而浆细胞可以依靠人体自身B细胞的不断再生得到弥补。以T细胞双特异性抗体产品为例，BCMA靶点的产品主要有再生元制药公司研发(Regeneron Pharmaceuticals,Inc.(REGN))的REGN5458，强生研发的Teclistama，以及安进的AMG420。REGN5458目前已结束临床一期实验(NCT03761108)。19名接受治疗的患者种，42％达到完全缓解(CR)或者严格完全缓解(sCR)；Teclistamab已进入临床二期实验(NCT04557098)，临床一期结果显示，总体响应率(ORR)为65％，40％的患者完全缓解(CR)；AMG420的临床一期(NCT03836053)显示，总体响应率(ORR)为71％ [2],[3]。
百时美施贵宝(BMS)研发的针对多发性骨髓瘤BCMA靶点的CAR-T产品－bb2121在2021年5月被FDA批准为第一款多发性骨髓瘤上市的CAR-T产品。该产品总体响应率(ORR)为72％，28％患者可达到严格完全缓解(sCR) [4]。尽管该产品为多发性骨髓的治愈带来了希望，但是从公布的数据来看，其完全缓解(CR)的患者中22个月疾病无进展生存期占比也不到50％，可见治疗后期也存在较高的复发率。此外，由于该疗法清除所有的浆 细胞，仍然有血细胞减少免疫球蛋白降低等引发的副作用。GPRC5D表达相对BCMA更为特异，仅在骨髓瘤患者的浆细胞中表达，而正常组织几乎不表达，仅在毛囊组织中能检测到明显的RNA和蛋白表达 [5],[6]。在敲除GPRC5D的老鼠与正常野生型的表型比较上(包括体重，器官形态学差异，生殖率等)，也未发现明显差异 [7]，可见GPRC5D缺失对生存和正常器官的生长发育和代谢并非必需，其清除的副作用较小。进一步发现，BCMA的表达和GPRC5D并没有相关性，两者虽然都同时在浆细胞中表达，但表达谱相对独立。可见对于BCMA CAR-T疗法后期患者中BCMA表达偏低，也可用靶向GPRC5D来治疗。
目前在研的靶向GPRC5D产品主要有两款，双抗有强生公司开发的GPRC5D/CD3的双抗Talquemab也处于临床1/2期研究中，中等剂量的响应率为70％，其中65％的三重耐药者有响应，83％的五重耐药者有响应 [6]。CAR-T产品主要有优瑞科公司研发的MCARH109，处于临床一期试验中，2021ASH年会报道的数据显示12名接受GPRC5D CAR-T注射液的患者，3/12为PR部分缓解，3/12为VGPR非常好部分缓解，2/12为sCR严格完全缓解(sCR) [5]。
医疗市场上仍需要新的结构简单且免疫原性低的CAR-T产品。
发明内容
在一方面，本文提供了嵌合抗原受体(CAR)，其胞外抗原结合结构域包括一个或更多个靶向GPRC5D的抗体分子或其抗原结合片段，所述抗体分子的重链可变区的HCDR1、HCDR2和HCDR3选自如下组合之一：
1)HCDR1的序列为GGSFSGYY(SEQ ID NO：1)；
HCDR2的序列为INHSGST(SEQ ID NO：2)；
HCDR3的序列为ARARRYGGRTRFDP(SEQ ID NO：3)；
2)HCDR1的序列为GFIFSSYG(SEQ ID NO：4)；
HCDR2的序列为ISSSGDYT(SEQ ID NO：5)；
HCDR3的序列为ARMSFRRYDH(SEQ ID NO：6)；以及
3)HCDR1的序列为GFSFSGYI(SEQ ID NO：7)；
HCDR2的序列为TSSSGTET(SEQ ID NO：8)；
HCDR3的序列为ARYYSKYGRSYHVDS(SEQ ID NO：9)。
在一些实施方案中，所述胞外抗原结合结构域包括串联连接的多个(如两个)所述抗体分子或其抗原结合片段，优选地，所述抗体分子或其抗原结合片段通过连接肽相连。
在一些实施方案中，所述两个所述抗体分子或其抗原结合片段相同或不同。
在一些实施方案中，所述抗体分子为单域抗体。
在一些实施方案中，所述抗体分子为全人源单域抗体。
在一些实施方案中，所述重链可变区(或所述单域抗体)包括SEQ ID NO：10、11或12所示的氨基酸序列；或者所述重链可变区包括与SEQ ID NO：10、11或12所示序列有至少90％、91％、92％、93％、94％、95％、96％、97％、98％、或99％序列一致性的氨基酸序列并且能够特异性结合GPRC5D。
在一些实施方案中，所述CAR从N端到C端依次包括所述胞外抗原结合结构域、跨膜结构域和胞内信号传导结构域。
在一些实施方案中，所述CAR在N末端还包括信号肽。
在一些实施方案中，所述信号肽包括SEQ ID NO：17所示的氨基酸序列。
在一些实施方案中，所述CAR在所述胞外抗原结合结构域和所述跨膜结构域之间还包括铰链区。
在一些实施方案中，所述铰链区包括SEQ ID NO：19所示的氨基酸序列。
在一些实施方案中，所述跨膜区包括SEQ ID NO：21所示的氨基酸序列。
在一些实施方案中，所述胞内信号传导结构域包括4-1BB胞内结构域和CD3ζ胞内结构域。
在一些实施方案中，所述4-1BB胞内结构域包括SEQ ID NO：23所示的氨基酸序列。
在一些实施方案中，所述CD3ζ胞内结构域包括SEQ ID NO：25所示的氨基酸序列。
在一些实施方案中，所述CAR的末端还连接有用于CAR-T细胞清除的安全开关。
在一些实施方案中，在所述CAR的C末端连接有安全开关。
在一些实施方案中，所述安全开关包括：tEGFR，或具有自杀能力的融合蛋白。
在一些实施方案中，所述CAR的C末端连接有截短形式的EGFR分子(tEGFR)。
在一些实施方案中，在tEGFR的N末端还连接有CSF2RA信号肽。
在一些实施方案中，所述tEGFR包括SEQ ID NO：31所示的氨基酸序列。
在一些实施方案中，所述CSF2RA信号肽包括SEQ ID NO：29所示的氨基酸序列。
在一些实施方案中，所述安全开关通过自裂解多肽与CAR相连。
在一些实施方案中，所述自裂解多肽包括SEQ ID NO：27或33所示的氨基酸序列。
在一些实施方案中，所述CAR具有SEQ ID NO：35、37或39所示的氨基酸序列；或者所述CAR的氨基酸序列与SEQ ID NO：35、37或39所示序列有至少90％、91％、92％、93％、94％、95％、96％、97％、98％、或99％的序列一致性。
另一方面，本文提供了双特异性嵌合抗原受体(CAR)，其胞外抗原结合结构域包括(i)靶向GPRC5D的抗体分子或其抗原结合片段和(ii)靶向第二靶点的抗体分子或其抗原结合片段，所述靶向GPRC5D的抗体分子的单域抗体重链可变区(V HH)及全抗scFv(可变区)如上所述，所述第二抗体分子选自CD3、BCMA或其组合。
另一方面，本文还提供了分离的核酸分子，其编码上述CAR。
在一些实施方案中，所述核酸分子包括SEQ ID NO：13、14、15、16、18、20、22、24、26、28、30、32、34、36和38任一项所示的核苷酸序列。
另一方面，本文还提供了表达载体，其包括上述核酸分子。
在一些实施方案中，所述表达载体选自质粒、逆转录病毒载体和慢病毒载体。
另一方面，本文还提供了宿主细胞，其包括上述CAR、核酸分子或表达载体。
在一些实施方案中，所述宿主细胞为免疫细胞。
在一些实施方案中，所述宿主细胞包括T细胞或NK细胞，优选293T细胞。
另一方面，本文还提供了工程化免疫细胞，其表达上述CAR。
在一些实施方案中，所述工程化免疫细胞包括免疫细胞，优选T细胞或NK细胞。
在一些实施方案中，所述工程化免疫细胞包括自体细胞或异体细胞。
另一方面，本文还提供了药物组合物，其包括上述细胞以及药学上可接受的载体。
另一方面，本文还提供了上述CAR、核酸分子、表达载体、宿主细胞、或工程化免疫细胞在制备用于预防或治疗GPRC5D相关疾病的药物中的用途。
在一些实施方案中，所述GPRC5D相关疾病为癌症或自身免疫疾病，所述癌症优选浆细胞恶性肿瘤疾病，例如多发性骨髓瘤；或者B细胞恶性疾病，例如霍奇金淋巴瘤或非霍奇金淋巴瘤。
另一方面，本文还提供了预防或治疗GPRC5D相关疾病的方法，包括以治疗有效量的上述工程化免疫细胞或药物组合物向有需要的受试者给药。
在一些实施方案中，所述方法还包括以EGFR抗体向所述受试者给药来抑制所述工程化免疫细胞或所述药物组合物的活性。
在一些实施方案中，所述GPRC5D相关疾病为癌症或自身免疫疾病，所述癌症优选浆细胞恶性肿瘤疾病，例如多发性骨髓瘤；或者B细胞恶性疾病，例如霍奇金淋巴瘤或非霍奇金淋巴瘤。
本申请基于GPRC5D的极特异的表达谱，潜在的安全性和BCMA疗法已取得良好的疗效，开发了一款靶向GPRC5D的CAR-T产品，用于治疗难治复发性多发性骨髓瘤，其GPRC5D结合域开发来源于人源化单域抗体库筛选，结构相对简单，且免疫原性低。
附图说明
图1显示了GPRC5D CAR载体构建的结构示意图。CAR包括胞外信号肽(SP)，与靶抗原GPRC5D结合区的ScFv(VL-linker-VH)或Single domain(V HH)，细胞膜和细胞外结合区之间的铰链区和跨膜区(CD8a hinge+TM)，4-1BB共刺激分子，CD3ζ胞内结构域，T2A自裂解肽，截短的tEGFR。
图2显示了各组CAR-T细胞的tEGFR的表达情况。
图3A-3C显示了各组CAR-T细胞对多种靶细胞(8226-luc、U266-luc、MM1.s-luc、CCRF-luc、Raji-luc)的杀伤结果。
图4显示了多次靶细胞(8226)刺激后各种CAR-T的体外增殖情况。
图5显示了不同组织来源肿瘤细胞对各组CAR-T细胞的CD107a脱粒作用结果。
具体实施方式
除非另有说明，本文使用的所有技术和科学术语具有本领域普通技术人员所通常理解的含义。
抗体指由浆细胞(效应B细胞)分泌、被机体免疫系统用来中和外来物质(多肽、病毒、细菌等)的免疫球蛋白。该外来物质相应地称作抗原。经典抗体分子的基本结构是由2个相同重链和2个相同轻链组成的4聚体。根据氨基酸序列的保守性差异，将重链和轻链分为位于氨基端的可变区(V)和位于羧基端的恒定区(C)。一条重链和一条轻链的可变区相互作用形成了抗原结合部位(Fv)。在可变区中，某些区域氨基酸残基的组成和排列次序比可变区内的其它区域(框架区，FR)更易变化，称为高变区(HVR)，高变区实际上是抗体与抗原结合的关键部位。由于这些高变区序列与抗原决定簇互补，故又称为互补决定区(complementarity-determining region,CDR)。重链和轻链均具有三个互补决定区，分别称为HCDR1、HCDR2、HCDR3和LCDR1、LCDR2、LCDR3。
抗体分子的“抗原结合片段”指抗体分子中参与抗原特异性结合的氨基酸片段，例如，Fab、Fab’和(Fab’) 2等。具有抗原结合能力的单链抗体和单域抗体为单肽链抗体分子，可视为经典抗体分子的“抗原结合片段”。
单链抗体(single chain fragment variable,scFv)，是由抗体重链可变区和轻链可变区通过短肽连接成一条肽链而构成。通过正确折叠，来自重链和轻链的可变区通过非共价键相互作用形成Fv段，因而scFv能较好地保留其对抗原的亲和活性。
“单域抗体(single domain antibody，sdAb)”，或者也称为“V HH抗体”，指具有抗原结合能力，包括重链可变区而无轻链的抗体分子。从结构上看，单域抗体也可以认为是抗体分子的一种抗原结合片段。其首先在骆驼科动物中被发现，随后，研究人员通过抗体库(例如噬菌体展示文库)筛选发现了更多的具有抗原结合能力的单域抗体。单域抗体相对于普通抗体分子(例如，经典四聚体抗体分子)或其抗原结合片段具有一些优势，例如包括但不限于：分子量更小，使用于人体时易于到达普通抗体分子难以到达的组织或部位，或者，能够接触到蛋白或多肽中普通抗体分子难以接触到的抗原表位；更加稳定，能够耐受例如温度和pH的变化以及变性剂和蛋白酶的作用。
“全人源抗体”指可变区和恒定区(如果有的话)均衍生自人生殖系免疫球蛋白序列的抗体。全人源抗体可通过多种技术获得，包括噬菌体抗体库技术、单个B细胞克隆技术、转基因小鼠技术(例如，使用引入了人生殖系免疫球蛋白基因并去除了小鼠自身生殖系免疫球蛋白基因的转基因小鼠)等。相对于动物源抗体(例如鼠源抗体)，全人源抗体在用于人患者时具有免疫原性小、安全性高的优势。
“靶向”或“特异性结合”指，相对于环境中同时存在的其他分子，一种分子(例如抗体或其抗原结合片段)对另一种分子(如肿瘤细胞表面抗原)具有更高的结合亲和力。“靶向”或 “特异性结合”并不排除该分子可以对一种以上的分子具有结合亲和力，例如双特异性抗体可以对两种不同抗原具有高亲和力。
GPRC5D是G蛋白偶联受体C5家族亚型D，属于一种孤儿受体，为7次跨膜蛋白。孤儿受体(Orphan Receptor)是指与其它已确认的受体结构明显相似，但其内源配体还未发现的受体。GPRC5D在原代多发性骨髓瘤细胞表面高表达，而在正常组织的表达仅限于毛囊区域，有研究表明65％的多发性骨髓瘤患者GPRC5D有超过50％的表达阈值，凭借这一特点，GPRC5D成为了治疗多发性骨髓瘤(MM)的潜在靶标。
术语“多肽”和“蛋白质”可互换使用且指氨基酸残基的聚合物。氨基酸残基的此类聚合物可含有天然或非天然氨基酸残基且包括但不限于氨基酸残基构成的肽、寡肽、二聚体、三聚体和多聚体。全长蛋白与其片段皆涵盖于该定义中。该术语亦包括多肽的表达后修饰，例如糖基化、唾液酸化、乙酰化、磷酸化和类似修饰。此外，出于本发明的目的，“多肽”指一种蛋白质，其包括对天然序列的修饰，诸如缺失、添加和取代(实际上通常为保守的)，只要该蛋白质保持所需活性即可。这些修饰可为有目的的，如经由定点突变诱发；或可为偶然的，诸如经由产生蛋白质的宿主的突变或因PCR扩增所致的误差。
本文中，术语“核酸分子”、“核酸”和“多核苷酸”可互换使用，指核苷酸聚合物。此类核苷酸聚合物可含有天然和/或非天然核苷酸且包括(但不限于)DNA、RNA和PNA。“核酸序列”指包含于核酸分子或多核苷酸中的核苷酸线性序列。
术语“载体”指可经工程改造以含有目的多核苷酸(例如目的多肽的编码序列)的核酸分子或可在宿主细胞中复制的核酸分子(例如，核酸、质粒、或病毒等)。载体可包括以下组件中的一个或更多个：复制起点、一或更多个调控目的多核苷酸的表达的调控序列(诸如启动子和/或增强子子)和/或一个或更多个可选择标记物基因(诸如抗生素抗性基因和可用于比色分析中的基因，例如β-半乳糖)。术语“表达载体”指用于在宿主细胞中表达目的多肽的载体。
“宿主细胞”指可为或已为载体或经分离多核苷酸的接受体的细胞。宿主细胞可为原核细胞或真核细胞。示例性真核细胞包括哺乳动物细胞，诸如灵长类动物或非灵长类动物细胞；真菌细胞，诸如酵母；植物细胞；以及昆虫细胞。非限制性示例性哺乳动物细胞包括(但不限于)NSO细胞、293以及CHO细胞，以及其衍生细胞，诸如293-6E、CHO-DG44、CHO-K1、CHO-S和CHO-DS细胞。宿主细胞包括单个宿主细胞的后代，且后代可能由于自然、偶然或故意突变而不一定与原始母细胞完全一致(在形态或基因组DNA互补方面)。宿主细胞也包括在活体内经本文提供的核酸分子或表达载体转染的细胞。
“治疗”指对受试者进行处理以获得有益的或所期望的临床结果。本文所用的“治疗”涵盖各种处理手段，包括以任何可能的药物向受试者给药、手术、辐射等。出于本发明的目的，有益或所期望的临床结果包括但不限于以下的任一种或多种：减轻一种或更多种症状、减弱疾病程度、预防或延迟疾病扩散(例如转移，例如转移至肺或淋巴结)、预防或延迟疾病复发、延迟或减缓疾病进展、改善疾病病况、抑制疾病或疾病进展、阻滞其 发展和缓解(无论部分抑或完全缓解)。本文所提供的方法涵盖这些治疗方面中的任一种或多种。按照以上内容，“治疗”不需要完全去除病症或疾病的所有症状或完全缓解。
术语“治疗有效量”指足以在受试者体内引起临床医师所期望的生物学或医学反应的活性化合物的量。本发明融合蛋白的“治疗有效量”可由本领域技术人员根据给药途径、受试者的体重、年龄、病情等因素而确定。例如，典型的日剂量范围可以为每kg体重0.01mg至100mg或更多活性成分。
提及药物组合物，所使用的术语“药学上可接受的载体”指可以安全地进行施用的固体或液体稀释剂、填充剂、抗氧化剂、稳定剂等物质，这些物质适合于人和/或动物给药而无过度的不良副反应，同时适合于维持位于其中的药物或活性剂的活力。依照给药途径，可以施用本领域众所周知的各种不同的载体，包括，但不限于糖类、淀粉、纤维素及其衍生物、麦芽糖、明胶、滑石、硫酸钙、植物油、合成油、多元醇、藻酸、磷酸缓冲液、乳化剂、等渗盐水、和/或无热原水等。本文所提供的药物组合物可以制成粉末、注射剂等临床可接受的剂型。可以使用任何适当的途径向受试者施用本发明的药物组合物，例如可通过口服、静脉内输注、肌肉内注射、皮下注射、腹膜下、直肠、舌下，或经吸入、透皮等途径给药。
“受试者”指动物，例如哺乳动物，包括(但不限于)人类、啮齿动物、猿猴、猫科动物、犬科动物、马科动物、牛科动物、猪科动物、绵羊、山羊、哺乳类实验动物、哺乳类农畜、哺乳类运动动物和哺乳类宠物。受试者可为雄性或雌性且可为任何适龄受试者，包括婴儿、幼年、青年、成年和老年受试者。在一些实例中，受试者指需要治疗疾病或病症的个体。在一些实例中，接受治疗的受试者可为患者，其患有与该治疗有关联的病症，或有风险患上该病症。在特定实例中，受试者为人类，诸如人类患者。该术语通常可与“患者”、“检测对象”、“治疗对象”等互换使用。
提及氨基酸或核苷酸序列时，术语“序列一致性(sequence identity)”(也称为“序列同一性”)指两氨基酸或核苷酸序列(例如查询序列和参照序列)之间一致性程度的量，一般以百分比表示。通常，在计算两氨基酸或核苷酸序列之间的一致性百分比之前，先进行序列比对(alignment)并引入缺口(gap)(如果有的话)。如果在某个比对位置，两序列中的氨基酸残基或碱基相同，则认为两序列在该位置一致或匹配；两序列中的氨基酸残基或碱基不同，则认为在该位置不一致或错配。在一些算法中，用匹配位置数除以比对窗口中的位置总数以获得序列一致性。在另一些算法中，还将缺口数量和/或缺口长度考虑在内。出于本发明的目的，可以采用公开的比对软件BLAST(可在网页ncbi.nlm.nih.gov找到)，通过使用缺省设置来获得最佳序列比对并计算出两氨基酸或核苷酸序列之间的序列一致性。
嵌合抗原受体(CAR)，也称为嵌合T细胞受体、嵌合免疫受体，为一种工程化的蛋白受体分子，其可将期望的特异性赋予免疫效应细胞，例如与特定肿瘤抗原结合的能力。嵌合抗原受体通常由胞外抗原结合结构域、跨膜结构域和胞内信号结构域构成。多数情 况下，抗原结合结构域为一段scFv序列，负责识别和结合特定的抗原。胞内信号结构域通常包括免疫受体酪氨酸活化基序(ITAM)，例如来源于CD3ζ分子的信号传导结构域，负责激活免疫效应细胞，产生杀伤作用。另外，嵌合抗原受体还可在氨基端包括负责新生蛋白在细胞内定位的信号肽，以及在抗原结合结构域和跨膜结构域之间包括铰链区。除了信号传导结构域，胞内信号结构域还可包括例如来源于4-1BB分子的共刺激结构域。
“CAR-T细胞”指表达CAR的T细胞，通常采用编码CAR的表达载体转导T细胞获得。常用的表达载体为病毒载体，例如慢病毒表达载体。经嵌合抗原受体修饰的T细胞(CAR-T)不受主要组织相容性复合体的限制，具有特异性靶向杀伤活性和持久扩增的能力。除T细胞外，也可以用编码CAR的表达载体转化诸如NK细胞的其他淋巴细胞，获得表达该CAR的靶向杀伤性细胞。
“自剪切肽”指可经核糖体跳跃而非蛋白酶水解来实现剪切蛋白的功能的短肽，可包括T2A、F2A和P2A等。
“CSF2RA信号肽”，即集落刺激因子2受体α亚基(colony stimulating factor 2 receptor subunit alpha)信号肽，可引导新合成的蛋白如EGFRt在CAR-T细胞表面表达。
“EGFRt”或“tEGFR”在本文中可以互换使用，指编码截短的人表皮生长因子受体多肽的基因或其编码产物，其缺乏远端膜EGF结合域和细胞质信号传导尾，但保留了由抗EGFR抗体识别的细胞外表位。EGFRt可用作具有遗传修饰细胞功能的非免疫原性选择工具以及追踪标记。在本文中，其一方面可作为CAR-T细胞的标记分子，另一方面还可以在需要时用于清除体内的CAR-T细胞，例如通过EGFR抗体(例如，西妥昔单抗)介导的ADCC途径(参见US8802374B2)，即在临床转化时作为安全开关使用。
除了使用tEGFR外，本文提供的CAR-T细胞还可以考虑与其他机制的安全开关联合使用。例如，可以对本文提供的CAR进行加工以使其可以利用二聚化化学诱导剂(CID)来控制CAR活性。在一个实例中，可以将本文提供的完整CAR以分开的融合蛋白进行表达(例如将CD3ζ胞内结构域与其他部分分开表达)，并利用基于FKBP/FRB的结合结构域和雷帕霉素(或雷帕霉素类似物)的二聚化机制在细胞内重建完整CAR，从而可通过雷帕霉素的存在与否来控制CAR的完整性和活性(例如，参见美国专利11,084,880中的描述)。另外，当采用自剪切肽使得能够同时表达具有自杀能力的融合蛋白的时，可以通过降解CAR分子或者导致CAR-T细胞死亡来控制CAR-T细胞活性。这方面的实例包括使用PROTAC技术降解CAR分子以及使用具有诱导细胞凋亡能力的蛋白，如caspase-9 [8]。
氨基酸和核苷酸序列简要说明
以下提供对本文中(尤其是实施例中)使用到的一些抗体或抗原结合片段(如单域抗体)和CAR以及相关多肽的氨基酸和编码核酸序列的说明。
SEQ ID NO：1为#18克隆的CDR1的氨基酸序列，即GPRC5D CAR 2290的胞外抗原结合结构域的CDR1。
SEQ ID NO：2为#18克隆的CDR2的氨基酸序列，即GPRC5D CAR 2290的胞外抗原结合结构域的CDR2。
SEQ ID NO：3为#18克隆的CDR3的氨基酸序列，即GPRC5D CAR 2290的胞外抗原结合结构域的CDR3。
SEQ ID NO：4为#39克隆的CDR1的氨基酸序列，即GPRC5D CAR 2436的胞外抗原结合结构域的CDR1。
SEQ ID NO：5为#39克隆的CDR2的氨基酸序列，即GPRC5D CAR 2436的胞外抗原结合结构域的CDR2。
SEQ ID NO：6为#39克隆的CDR3的氨基酸序列，即GPRC5D CAR 2436的胞外抗原结合结构域的CDR3。
SEQ ID NO：7为#41克隆的CDR1的氨基酸序列，即GPRC5D CAR 2438的胞外抗原结合结构域的CDR1。
SEQ ID NO：8为#41克隆的CDR2的氨基酸序列，即GPRC5D CAR 2438的胞外抗原结合结构域的CDR2。
SEQ ID NO：9为#41克隆的CDR3的氨基酸序列，即GPRC5D CAR 2438的胞外抗原结合结构域的CDR3。
SEQ ID NO：10为#18克隆的V HH的氨基酸序列，即GPRC5D CAR 2290的胞外抗原结合结构域的序列。
SEQ ID NO：11为#39克隆的V HH的氨基酸序列，即GPRC5D CAR 2436的胞外抗原结合结构域的序列。
SEQ ID NO：12为#41克隆的V HH的氨基酸序列，即GPRC5D CAR 2438的胞外抗原结合结构域的序列。
SEQ ID NO：13为#18克隆的V HH的编码序列，即GPRC5D CAR 2290的胞外抗原结合结构域的编码序列。
SEQ ID NO：14为#39克隆的V HH的编码序列，即GPRC5D CAR 2436的胞外抗原结合结构域的编码序列。
SEQ ID NO：15为#41克隆的V HH的编码序列，即GPRC5D CAR 2438的胞外抗原结合结构域的编码序列。
SEQ ID NO：16为CD8α信号肽的编码核酸序列。CD8α信号肽可用于将新合成的CAR引导到细胞表面。成熟的CAR中该信号肽可被切除，因而可不包括该信号肽。
SEQ ID NO：17为CD8α信号肽的氨基酸序列。
SEQ ID NO：18为CD8α铰链区的编码核酸序列。CD8α铰链区可用于在CAR中连接胞外抗原结合结构域和跨膜结构域。
SEQ ID NO：19为CD8α铰链区的氨基酸序列。
SEQ ID NO：20为CD8α跨膜区的编码核酸序列。
SEQ ID NO：21为CD8α跨膜区的氨基酸序列。
SEQ ID NO：22为4-1BB胞内结构域的编码核酸序列。
SEQ ID NO：23为4-1BB胞内结构域的氨基酸序列。
SEQ ID NO：24为CD3ζ胞内信号传导结构域的编码核酸序列。
SEQ ID NO：25为CD3ζ胞内信号传导结构域的氨基酸序列。
SEQ ID NO：26为自剪切肽T2A的编码核酸序列。
SEQ ID NO：27为自剪切肽T2A的氨基酸序列。由于自剪切肽的存在，导致自剪切肽两侧的蛋白(CAR和EGFRt)在翻译过程中就彼此分开。为了描述方便，可认为作为融合蛋白的包括多个元件的CAR也可包括T2A以及EGFRt。
SEQ ID NO：28为CSF2RA信号肽的编码核酸序列。
SEQ ID NO：29为CSF2RA信号肽的氨基酸序列。可位于EGFRt上游用于将新合成的EGFRt引导到细胞表面。CSF2RA信号肽可被切除而离开EGFRt。
SEQ ID NO：30为EGFRt的编码核酸序列。
SEQ ID NO：31为EGFRt的氨基酸序列。
SEQ ID NO：32为自剪切肽P2A的编码核酸序列，也可以选择自剪切肽P2A替代T2A作为CAR和EGFRt之间的连接肽。
SEQ ID NO：33为自剪切肽P2A的氨基酸序列。
SEQ ID NO：34为GPRC5D CAR 2290(#18)的编码序列。
SEQ ID NO：35为GPRC5D CAR 2290(#18)的氨基酸序列。
SEQ ID NO：36为GPRC5D CAR 2436(#39)的编码序列。
SEQ ID NO：37为GPRC5D CAR 2436(#39)的氨基酸序列。
SEQ ID NO：38为GPRC5D CAR 2438(#41)的编码序列。
SEQ ID NO：39为GPRC5D CAR 2438(#41)的氨基酸序列。
其中，SEQ ID NO：34-39所示的序列仅为CAR结构的编码或氨基酸序列，不包含EGFRt元件。
此外，上述克隆#18、#39和#41为本申请提供的一些用于CAR构建的靶向GPRC5D的具体单域抗体的实例，用于构建CAR的克隆还包括单域抗体#14、#10、和#8，以及scFv#46、#47等。
嵌合抗原受体(CAR)
本文提供的嵌合抗原受体(CAR)，其是人工构建的嵌合蛋白，包括特异性结合GPRC5D的胞外抗原结合结构域、跨膜结构域、以及一个或更多个细胞内信号传导结构域。这些CAR的特征包括它们以非MHC限制的方式将T细胞特异性和反应性重定向至表达GPRC5D的细胞的能力。非MHC限制的GPRC5D识别使表达所公开的CAR的T细胞或NK细胞能够独立于抗原加工而识别抗原。
胞内信号传导结构域可以包括T细胞受体信号传导结构域、T细胞共刺激信号传导结构域或两者。T细胞受体信号传导结构域可包括T细胞受体的细胞内结构域，例如CD3ζ蛋白的细胞内部分。共刺激信号传导结构域为共刺激分子的细胞内结构域。共刺激分子是淋巴细胞对抗原的有效反应所需的抗原受体或其配体以外的细胞表面分子。
1.胞外抗原结合结构域
一些实施方案中，在本文提供的CAR包括与GPRC5D特异性结合的胞外抗原结合结构域。例如，抗原结合结构域可以是scFv，其可与GPRC5D特异性结合的通过肽接头连接的任何抗体或其抗原结合片段的重链可变区和轻链可变区。又例如，对于利用特异性结合GPRC5D的单域抗体构建CAR，胞外抗原结合结构域可包括来自该单域抗体的V HH，而不包括任何轻链可变区。
在一些实施方案中，本文提供的CAR的胞外抗原结合结构域包括来自#18克隆的V HH，上述#18V HH的HCDR1、HCDR2和HCDR3的序列如下：
HCDR1的序列为GGSFSGYY(SEQ ID NO：1)；
HCDR2的序列为INHSGST(SEQ ID NO：2)；
HCDR3的序列为ARARRYGGRTRFDP(SEQ ID NO：3)。
在一些实施方案中，本文提供的CAR的胞外抗原结合结构域包括来自#39克隆的V HH，上述#39V HH的HCDR1、HCDR2和HCDR3的序列如下：
HCDR1的序列为GFIFSSYG(SEQ ID NO：4)；
HCDR2的序列为ISSSGDYT(SEQ ID NO：5)；
HCDR3的序列为ARMSFRRYDH(SEQ ID NO：6)。
在一些实施方案中，本文提供的CAR的胞外抗原结合结构域包括来自#18克隆的V HH，上述#41V HH的HCDR1、HCDR2和HCDR3的序列如下：
HCDR1的序列为GFSFSGYI(SEQ ID NO：7)；
HCDR2的序列为TSSSGTET(SEQ ID NO：8)；
HCDR3的序列为ARYYSKYGRSYHVDS(SEQ ID NO：9)。
在一些实施方案中，本文提供的CAR的胞外抗原结合结构域包括来自#18克隆的V HH(SEQ ID NO：10)或其任何功能性变体。
在另一些实施方案中，本文提供的CAR胞外抗原结合结构域包括来自#39克隆的V HH(SEQ ID NO：11)或其任何功能性变体。
在另一些实施方案中，本文提供的CAR胞外抗原结合结构域包括来自#41克隆的V HH(SEQ ID NO：12)或其任何功能性变体。
这里的功能性变体可以与其亲本序列(本文中提供的VH氨基酸序列)相比，包括数个个氨基酸残基的插入、缺失或替换，并且基本上保留了特异性结合GPRC5D的能力。
在一些实施方案中，功能性变体可以与其亲本序列相比，可包括1、2、3、4、5、6、7、8、9、10、11、12、13、14、15、16、17、18、19、20、21、22、23、24、25、26、 27、28、29、30或更多个氨基酸残基的插入、缺失或替换，同时保留了亲本序列与GPRC5D的结合亲和力的至少50％、60％、70％、80％、90％，或甚至高于亲本序列。本领域技术人员已知，VH框架区序列可以有相当大的变动余地，只要其可以维持相应CDR序列的空间位置。例如，可改变VH氨基酸序列中的框架区序列，例如以不同种属抗体分子的框架区互相替换，可获得基本上保留了抗原结合能力的功能性变体。此外，还可以通过改变少数几个CDR序列中的残基，例如1个、2个或3个氨基酸残基，并检测改动后的抗体分子与相应抗原的结合能力，来获得依然具有抗原结合能力的功能性变体。
功能相似氨基酸的保守氨基酸替换是本领域普通技术人员熟知的。以下六组是被认为是彼此保守替换的氨基酸的实例：
1)丙氨酸(A)；丝氨酸(S)；苏氨酸(T)；
2)天冬氨酸(D)，谷氨酸(E)；
3)天冬酰胺(N)，谷氨酰胺(Q)；
4)精氨酸(R)，赖氨酸(K)；
5)异亮氨酸(I)，亮氨酸(L)，蛋氨酸(M)，缬氨酸(V)；和
6)苯丙氨酸(F)，酪氨酸(Y)，色氨酸(W)。
本文提供的CAR的胞外抗原结合结构域可包括两个或多个具有GPRC5D结合能力的上文所述V HH片段。它们直接可直接串联连接或通过肽接头串联连接。采用两个或多个串联连接的V HH片段作为胞外抗原结合结构域或其部分，有助于促进构建的CAR对靶分子(例如GPRC5D)的识别和结合。优选地，这些VH片段分别结合GPRC5D上的不同表位。
CAR可以包括信号肽序列，例如，位于抗原结合结构域的N端。可以使用任何合适的信号肽序列。在一个实施方案中，信号肽序列是CD8α信号肽。虽然信号肽序列可以促进CAR在细胞表面表达，但在表达的CAR中信号肽序列的存在对于CAR发挥功能并不是必需的。当CAR在细胞表面表达后，信号肽序列可能会从CAR上切下。因此，在一些实施方案中，CAR缺乏信号肽序列。
在CAR的抗原结合结构域和跨膜结构域之间，可能存在铰链区(或间隔结构域)，其包括多肽序列。铰链区可包含多达300个氨基酸，优选10至100个氨基酸。在一个实施方案中，本文提供的CAR包括CD8α蛋白铰链区。
2.跨膜结构域
CAR可以包括与CAR的胞外结构域融合的跨膜结构域。在一个实施方案中，使用与CAR中其他元件(如交联区或胞内信号传导结构域)天然相关的跨膜结构域。
跨膜结构域可以源自天然或合成来源。在来源是天然的情况下，该结构域可以源自任何膜结合或跨膜蛋白。用在所公开的CAR中的示例性跨膜结构域可以至少包括T细胞受体的α、β或ζ链、CD28、CD3ε、CD45、CD4、CD5、CDS、CD9、CD 16、CD22、CD33、CD37、CD64、CD80、CD86、CD 134、CD137、CD154的跨膜结构域。或者，跨膜结构域可以是合成的，在这种情况下，它将主要包含疏水残基，例如亮氨酸和缬氨 酸。在一些实施方案中，苯丙氨酸、色氨酸和缬氨酸的三联体将出现在合成跨膜结构域的每一端。
任选地，优选长度为2至10个氨基酸的短寡肽或多肽接头可以形成跨膜结构域与CAR的细胞内T细胞信号传导结构域和/或T细胞共刺激结构域之间的连接。示例性接头序列包括一个或更多个甘氨酸-丝氨酸双联体。
在一些实施方案中，跨膜结构域包括T细胞受体的跨膜结构域，例如CD8α跨膜结构域(SEQ ID NO：21)。
3.胞内结构域
CAR的细胞内区域包括一个或更多个胞内T细胞信号传导结构域，负责激活表达CAR的T细胞的至少一种正常效应功能。示例性T细胞信号传导结构域在本文中有提供，并且是本领域普通技术人员已知的。
虽然在CAR中可以使用整个细胞内T细胞信号传导结构域，但在许多情况下，没有必要使用整个链。就使用细胞内T细胞信号传导结构域的截短部分而言，该截短部分可代替完整链使用，只要它转导相关的T细胞效应器功能信号即可。
用在CAR中的细胞内T细胞信号传导结构域的例子包括T细胞受体(TCR)的细胞质序列和协同作用以在抗原受体结合后启动信号转导的共刺激分子，以及这些序列的任何衍生物或变体和任何具有相同功能的合成序列。
T细胞受体信号传导结构域以刺激方式或抑制方式调节T细胞受体复合物的激活。本文所公开的CAR可以包括以刺激方式起作用的细胞质信号传导序列，其可以包含被称为基于免疫受体酪氨酸的激活基序或ITAM的信号传导基序。可包含在所公开的CAR中的含有ITAM的初级细胞质信号传导序列的实例包括来自CD3ζ、FcRγ、FcRβ、CD3γ、CD3δ、CD3ε、CDS、CD22、CD79a、CD79b和CD66d蛋白的胞内结构域。在一些实施方案中，CAR中的细胞质信号传导分子包括来自CD3ζ的细胞内T细胞信号传导结构域。
本文提供的CAR的胞内结构域可以包括CD3ζ链部分和细胞内共刺激信号传导结构域。该共刺激信号传导结构域可包括共刺激分子的细胞内结构域。共刺激分子是淋巴细胞对抗原的有效反应所需的抗原受体或其配体以外的细胞表面分子。此类分子的实例包括CD27、CD28、4-1BB(CD137)、OX40(CD134)、CD30、CD40、PD-1、ICOS、淋巴细胞功能相关抗原1(LFA-1)、CD2、CD7、LIGHT、NKG2C和B7-H3。。
在一些实施方案中，CAR可以包括CD3ζ信号传导结构域、CD8信号传导结构域、CD28信号传导结构域、4-1BB信号传导结构域或其两种或更多种的组合。在一个实施方案中，胞内结构域包括CD3-ζ的信号传导结构域和CD28的信号传导结构域。在另一个实施方案中，细胞质结构域包括CD3ζ的信号传导结构域和4-1BB的信号传导结构域。在又一个实施方案中，细胞质结构域包括CD3-ζ的信号传导结构域以及CD28和CD137的信号传导结构域。本领域普通技术人员可以根据需要改变CAR上一个或更多个T细胞信号传导结构域的顺序。本发明的CAR的细胞质信号传导部分内的细胞质信号传导序列可 以以随机或指定的顺序相互连接。任选地，短的，优选长度在2和10个氨基酸之间的多肽接头，可以形成该连接。甘氨酸-丝氨酸双联体提供了特别适合的接头。此外，在CAR的信号传导结构域和跨膜结构域之间，可以存在间隔结构域，其包括多肽序列。间隔结构域可包含多达300个氨基酸，优选10至100个氨基酸，最优选25至50个氨基酸。
4.CAR的额外描述
基于本文提供的CAR(作为亲本CAR)，本领域技术人员可对其进行改动，例如，将其与其他多肽融合，或者仅保留本文提供的CAR的片段，而这些融合分子或片段具有所述亲本CAR的至少部分生物活性，例如识别靶细胞(例如表达GPRC5D的肿瘤细胞)或检测、治疗或预防疾病的活性。
本文提供的CAR还可以在氨基或羧基末端，或在两个末端包括附加氨基酸，这些附加氨基酸不存在于亲本CAR的氨基酸序列中。理想地，附加氨基酸不干扰CAR或功能部分的生物学功能，例如识别靶细胞、检测癌症、治疗或预防癌症等。更理想地，与亲本CAR的生物学活性相比，附加氨基酸增强生物学活性。
还提供了本文描述的CAR的功能性变体，其与亲本CAR具有实质或显著的序列一致性或相似性，该功能变体保留了其作为变体的CAR的生物学活性。功能性变体涵盖例如本文描述的CAR(亲本CAR)的那些变体，其保留与亲本CAR相似程度、相同程度或更高程度识别靶细胞的能力。参考亲本CAR，功能性变体在氨基酸序列上与亲本CAR例如可以有至少约30％、约50％、约75％、约80％、约85％、约90％、约91％、约92％、约93％、约94％、约95％、约96％、约97％、约98％、约99％或更高一致性。
功能性变体例如可以包括具有至少一个保守氨基酸替换的亲本CAR的氨基酸序列。或者或另外，功能变体可包括具有至少一个非保守氨基酸替换的亲本CAR的氨基酸序列。在这种情况下，优选非保守氨基酸替换不干扰或抑制功能变体的生物学活性。非保守氨基酸替换可以增强功能变体的生物活性，使得功能变体的生物活性与亲本CAR相比增加。
本文提供的CAR可以包括合成氨基酸来代替一种或更多种天然存在的氨基酸。此类合成氨基酸是本领域已知的，包括例如氨基环己烷羧酸、正亮氨酸、a-氨基正癸酸、高丝氨酸、S-乙酰氨基甲基-半胱氨酸、反式-3-和反式-4-羟脯氨酸、4-氨基苯丙氨酸、4-硝基苯丙氨酸、4-氯苯丙氨酸、4-羧基苯丙氨酸、β-苯基丝氨酸、β-羟基苯丙氨酸、苯基甘氨酸、α-萘丙氨酸、环己基丙氨酸、环己基甘氨酸、二氢吲哚-2-羧酸、1,2,3,4-四氢异喹啉-3-羧酸、氨基丙二酸、氨基丙二酸单酰胺、N'-苄基-N'-甲基-赖氨酸、N',N'-二苄基-赖氨酸、6-羟基赖氨酸、鸟氨酸、α-氨基环戊烷羧酸、α-氨基环己烷羧酸、oc-氨基环庚烷羧酸、-(2-氨基-2-降冰片烷)-羧酸、γ-二氨基丁酸、α,β-二氨基丙酸、高苯丙氨酸和α-叔丁基甘氨酸等。
本文提供的CAR可以被糖基化、酰胺化、羧化、磷酸化、酯化、N-酰化、经由例如二硫键环化，或转化成酸加成盐和/或任选地二聚或多聚，或偶联。
产生嵌合抗原受体的方法、包括此类受体的T细胞及其用途(例如，用于治疗癌症)是本领域已知的。例如，编码所公开的嵌合抗原结合受体的核酸分子可以包括在表达载体中(例如慢病毒载体)用于在宿主细胞例如T细胞中表达，以制备所公开的CAR。在一些实施方案中，使用嵌合抗原受体的方法包括从受试者中分离T细胞，用编码嵌合抗原受体的表达载体(例如慢病毒载体)转化T细胞，并以表达嵌合抗原受体的工程化T细胞向受试者给药用于治疗，例如治疗受试者中的肿瘤。
核酸分子和表达
本文提供了编码构建CAR的各元件的编码核酸序列。这些核酸序列可以通过任何合适的方法制备，包括例如克隆、扩增或直接化学合成。化学合成产生单链寡核苷酸。这可以通过与互补序列杂交或通过使用单链作为模板以DNA聚合酶聚合而转化为双链DNA。技术人员会认识到，虽然DNA的化学合成通常限于约100个碱基的序列，但可以通过连接较短的序列来获得较长的序列。
示例性核酸可以通过克隆技术制备。适当的克隆和测序技术的例子，以及足以指导技术人员通过许多克隆练习的说明是已知的。核酸也可以通过扩增方法制备。扩增方法包括聚合酶链式反应(PCR)、连接酶链式反应(LCR)、基于转录的扩增系统(TAS)、自持序列复制系统(3SR)。多种克隆方法、宿主细胞和体外扩增方法是本领域技术人员熟知的。
在一些实施方案中，核酸分子编码本文提供的用于在T细胞中表达以产生嵌合抗原受体T细胞的CAR。编码嵌合抗原结合受体的核酸分子可以包含在载体(例如慢病毒载体)中以在宿主细胞例如T细胞中表达。示例性细胞包括T细胞、自然杀伤(NK)细胞、细胞毒性T淋巴细胞(CTL)和调节性T细胞。
核酸分子可以在重组工程细胞如细菌、植物、酵母、昆虫和哺乳动物细胞中表达。抗体、抗原结合片段可以表达为单独的V HH(对于单域抗体)、或V H和V L链或可以表达为融合蛋白。
为了产生scFv，可以将编码V H和V L的DNA片段可操作地连接到另一个编码柔性接头的片段，例如，编码氨基酸序列(Gly 4-Ser) 3，使得V H和V L序列可以表达为一种连续的单链蛋白，其V L和V H结构域通过该柔性接头连接
本领域技术人员熟悉可用于表达蛋白质的众多表达系统，包括大肠杆菌(E.coli)、其他细菌宿主、酵母和各种高等真核细胞，例如COS、CHO、HeLa和骨髓瘤细胞系。
为了产生CAR，宿主细胞优选哺乳动物细胞，例如人类细胞。在一些实施方案中，宿主细胞可以是外周血淋巴细胞(PBL)或外周血单个核细胞(PBMC)，或T细胞。T细胞可以是任何T细胞，例如培养的T细胞，例如原代T细胞，或来自培养的T细胞系的T细胞，例如Jurkat、SupTl等，或获得自哺乳动物的T细胞。如果从哺乳动物获得，T细胞可以从多种来源获得，包括但不限于血液、骨髓、淋巴结、胸腺或其他组织或体液。也 可以富集或纯化T细胞。T细胞可以是人T细胞。T细胞可以是从人分离的T细胞。T细胞可以是任何类型的T细胞并且可以处于任何发育阶段，包括但不限于CD4 +/CD8 +双阳性T细胞、CD4 +辅助T细胞，例如Th 1和Th 2细胞、CD8 +T细胞(例如，细胞毒性T细胞)、肿瘤浸润细胞、记忆T细胞、幼稚T细胞等。T细胞可以是CD8 +T细胞或CD4 +T细胞。
可以对编码本文描述的CAR或其元件的核酸进行修饰而不降低其生物活性。例如，可以进行一些修饰以促进克隆或表达。此类修饰是本领域技术人员熟知的并且包括例如终止密码子、在氨基末端添加以提供起始位点的甲硫氨酸、位于任一末端以产生方便定位的限制性位点的额外氨基酸(如聚组氨酸)，以有助于纯化步骤。除了重组方法之外，本公开的CAR或其元件也可以使用本领域众所周知的标准肽合成来全部或部分构建。
药物组合物和医疗用途
本文提供的CAR或表达CAR的宿主细胞(如T细胞或NK细胞)可特异性识别并结合表达GPRC5D的靶细胞，例如肿瘤细胞。
因此，在一些实施方案中，本文提供了用于预防或治疗与GPRC5D表达相关的疾病的药物组合物，其包括表达本文提供的CAR的宿主细胞，尤其是CAR-T细胞，以及药学上可接收的载体。这些药物组合物可配制适合于给药方式的剂型，例如注射液。
在另一些实施方案中，本文提供治疗与与GPRC5D表达相关的疾病的方法，其包括以有效量的本文提供的CAR-T细胞或包括CAR-T细胞的药物组合物向有需要的在受试者中给药。在一些实施方案中，给药方式为静脉内给药，例如静脉注射；肌肉内给药，例如肌肉注射；或者肿瘤部分原位给药等。
在另一些实施方案中，本文提供了上述CAR-T细胞在制备治疗与GPRC5D表达相关的疾病的药物中的用途。
在一些实施方案中，与GPRC5D表达相关的疾病的为肿瘤，例如浆细胞恶性肿瘤疾病(例如多发性骨髓瘤)或B细胞恶性疾病(例如霍奇金淋巴瘤或非霍奇金淋巴瘤)；或自身免疫疾病。
在一些实施方案中，本文CAR-T细胞或药物组合物可向受试者给药以预防或治疗肿瘤。在这些应用中，以治疗有效量的靶向表达GPRC5D的靶细胞的CAR-T细胞或药物组合物向受试者给药，从而减缓或抑制肿瘤的生长或转移，减少肿瘤体积，或抑制癌症的体征或症状。治疗有效量将取决于疾病的严重程度和患者的一般健康状况。治疗有效量是提供症状的主观缓解或临床医生或其他合格观察者所指出的客观可识别的改善的量。在一个实施方案中，治疗有效量是抑制肿瘤生长、抑制转移、减少肿瘤体积所必需的量，或可有效减少肿瘤的体征或症状的量。所施用的试剂的治疗有效量可以根据所期望效果和待治疗的受试者而变化。在一些实例中，治疗量是消除或减少患者的肿瘤负担、或预防或减少转移细胞增殖或减轻肿瘤症状的量。
任何给药方法都可用于所公开的CAR-T细胞或药物组合物，包括局部和全身给药。例如，可以使用局部、口服、血管内(如静脉内)、肌肉内、腹膜内、鼻内、皮内、鞘内和皮下给药。具体的给药方式和给药方案将由主治临床医生选择，考虑病例的具体情况(例如受试者、疾病、所涉及的疾病状态以及治疗是否是预防性的)。
本文提供的CAR-T细胞或药物组合物的给药可以伴随其他抗癌剂或抗血管生成剂给药或治疗性处理(例如肿瘤的手术切除或放射疗法)。例如，在以治疗有效量的CAR-T细胞给药之前或之后，受试者可以接受一种或更多种另外的疗法或治疗剂。例如，该另外的疗法可包括但不限于使用化疗剂、抗血管生成剂或其组合。在另一个实例中，在以有效量的本文提供的CAR-T细胞或药物组合物给药之前，通过手术或其他方式切除至少部分肿瘤或减小其大小或体积。
可以使用的另外的治疗剂的具体例子包括微管结合剂、DNA嵌入剂或交联剂、DNA合成抑制剂、DNA和RNA转录抑制剂、抗体、酶、酶抑制剂、基因调节剂和血管生成抑制剂。这些试剂(以治疗有效量给药)和治疗可以单独使用或组合使用。例如，任何合适的抗癌剂或抗血管生成剂可以与本文公开的CAR-T细胞或药物组合物联合给药。
CAR研究概述
本发明使用全人源噬菌体库进行抗体筛选，直接获得全人源的单克隆抗体。与传统杂交瘤技术相比，省却了困难的鼠源抗体人源化步骤，而且全人源抗体比人源化的鼠源抗体具有更低的免疫原性，在抗体药物或CAR-T开发上有更好的潜力。
通过全人源噬菌体库获得了特异性结合细胞表面GPRC5D抗原的抗体克隆(包括单域抗体#18、#39、#41、#14，#10、#8和scFv#46，#47等)，随后将这些克隆及对照抗体benchmark 1(可变区序列来自于中国专利申请公开CN 109715667 A的GC5B596)构建至二代CAR结构上，然后进行慢病毒包装，并转染T细胞，在CAR-T细胞水平，从靶细胞激活和杀伤，靶细胞刺激增殖等角度筛选出了功能强于对照benchmark1的全人源GPRC5D抗体克隆及候选CAR-T分子。
在开发流程上，通过噬菌体水平的抗体筛选/特异性鉴定，快速高效筛选到特异抗体克隆，后面直接衔接CAR-T功能测试，优选出最佳的候选抗体，优化了以CAR-T开发为目的的抗体筛选流程，在保证研究质量的同时，提高了研发效率。
下表列出了进行具体研究的部分CAR的重链可变区序列(V HH)以及用于CAR构建的其他元件的氨基酸序列和编码序列。
Figure PCTCN2022080836-appb-000001
Figure PCTCN2022080836-appb-000002
Figure PCTCN2022080836-appb-000003
Figure PCTCN2022080836-appb-000004
Figure PCTCN2022080836-appb-000005
Figure PCTCN2022080836-appb-000006
Figure PCTCN2022080836-appb-000007
Figure PCTCN2022080836-appb-000008
CAR-T细胞的体外功能验证
实验目的和原理：通过亲和淘选从噬菌体抗体库富集靶向GPRC5D蛋白的特异抗体克隆并进行筛选和鉴定后获得了具有和GPRC5D阳性靶细胞结合的克隆，但这些克隆构建至二代CAR上后，在CAR-T细胞上的功能需要进一步确认。为此，我们制备了这些克隆的慢病毒载体，并转导T细胞制备成CAR-T细胞。然后，通过CD107a脱粒实验(CD107a degranulation assay)和体外细胞杀伤实验(in vitro cytotoxicity assay)进行CAR-T细胞的体外生物学效力评估，并且将其与已发表的抗体序列(Benchmark 1)构成的CAR功能做了同步评估。通过这些CAR-T水平的功能验证，我们最终选择到了有效性和安全性都理想的候选单链抗体克隆，进行下游的CAR-T产品开发。
实施例
实施例1. CAR-T细胞表面EGFRt表达的检测
T细胞进行转染如图1所示结构的CAR慢病毒5～7天后检测EGFRt表达情况(图2)，共分为两批进行转染。CAR结构中包含CD8α信号肽、V HH或scFv、CD8α铰链区、CD8α跨膜区、4-1BB共刺激分子和CD3ζ，并用T2A连接一个截短的EFGR分子(EGFRt) 可在临床转化时作为安全开关使用。由于EGFRt与CAR分子共表达，所以可以作为CAR分子在T细胞表面分布的间接检测指标且不影响CAR的结构及功能。
CAR-T/EGFRt表达检测简要实验步骤如下：
1)取1×10 6CAR-T/T细胞每孔，加入PBS洗一遍，300g离心5分钟，弃上清。
2)用100μL PBS重悬细胞沉淀，分别加入5μL APC-CD5抗体及5μL APC-EGFR抗体，4℃避光孵育15分钟。
3)用PBS洗两遍，300g离心5分钟。
4)用200μL PBS重悬，流式上机检测。
主要材料和试剂：
APC anti-human EGFR Antibody,Clone AY13,BioLegend,Cat.No.352906；
胎牛血清(FBS),Gibco，Cat.No.10099141；
实验结果：
如图2所示，第七天检测，各组T细胞检测EGFR的表达，均≥30％。
实施例2. CD107a脱粒实验
实验原理和目的：
CD107a是细胞内微囊泡的标志物，当负载有颗粒酶的微囊泡与细胞膜融合后，细胞膜上的CD107a会增加，当用莫能酶素(monesin，购自BioLegend)阻断其回收时，可以定量反映微囊泡释放的强度。当CAR-T受到靶细胞上靶抗原刺激后，会造成颗粒酶释放，并可通过流式检测CD107a的增加来判断T细胞的激活情况。
CD107a脱粒简要实验步骤：
1)将待测的CAR-T细胞和靶细胞分别在室温下以300g离心5min，弃上清后，用1640培养基+10％FBS重悬为2x10 6个细胞/mL；
2)在96孔板中，分别加入100μL待测的CAR-T细胞和100μL靶细胞，并混匀；
3)在每孔细胞中加入1.5μL PE/Cy7anti-human CD107a抗体和0.2μL monensin，然后放入细胞培养箱中37℃，5％CO 2孵育4h；
4)孵育完成后，在4℃下300g离心5min后弃上清，用200μL PBS洗细胞2次；
5)用100μLPBS重悬细胞，并分别加入1.5μL BV421anti-human CD8和1.5μL APC anti-human EGFR抗体，混匀后在冰上避光孵育20min；
6)孵育完成后，用200μL PBS洗细胞3次；用100μLPBS重悬后，用流式细胞仪检测。
主要样品和试剂：
靶细胞U266-luc(GPRC5D+),8226-luci(GPRC5D+),MM.1S-luci(GPRC5D+)，Raji-luc(GPRC5D-),CCRF-luc(GPRC5D-),Molt4-luc(GPRC5D-),Jurkat-luc(GPRC5D-)；
胎牛血清(FBS),Gibco，Cat.No.10099141；
Monensin,BioLegend,Cat.No.420701；
PE/Cy7 mouse anti-human CD107a,BD,Cat.No.561348；
BV421 mouse anti-human CD8,BD,Cat.No.301036；
APC anti-human EGFR Antibody,Clone AY13,BioLegend,Cat.No.35290。
实验结果：
通过慢病毒转导的方式获得CAR-T细胞，将该CAR-T细胞在体外培养9-12天后进行CD107a脱粒实验。待检测的CAR-T细胞和靶细胞、莫能酶素和CD107a抗体共同孵育4h，CAR-T细胞与靶细胞的细胞密度均为2×10 5个细胞/mL。然后用CD8抗体、EGFR抗体标记样品后，进行流式检测。在Flowjo软件中分析，散点图中选取活细胞门(P1)，去除细胞碎片；在P1门中的细胞，经过分析选取单个分散细胞门(P2)；然后，在P2门中进一步选取CD8阳性的细胞(P3)；最后，在P3门中，分析EGFR抗体染色呈阳性的细胞(即CAR阳性细胞)中CD107a阳性的比例。分析结果如表1所示，GPRC5D CAR-T仅与GPRC5D阳性细胞共孵育后脱颗粒水平有明显升高，而与阴性的细胞系共孵育后无明显的脱颗粒水平升高。其中克隆号18、39、41的GPRC5D阳性细胞的脱颗粒水平较高，且均高于Benchmark 1。第二批，CAR细胞中同样的克隆号18和46，47的脱颗粒水平也高于Benchmark 1。14号的脱颗粒水平稍弱。第三批，CAR细胞中同样克隆号18和10，8的脱颗粒水平也高于Benchmark 1。
表1不同靶细胞对各组CAR-T细胞的CD107a脱粒作用结果。
Figure PCTCN2022080836-appb-000009
Figure PCTCN2022080836-appb-000010
Figure PCTCN2022080836-appb-000011
实施例3. 体外细胞杀伤实验
实验目的和原理：
体外细胞杀伤实验采用U266 MM1.s 8226作为GPRC5D的阳性靶细胞，Raji和CCRF细胞作为GPRC5D阴性靶细胞，进行GPRC5D CAR-T细胞的抗原特异性杀伤能力评价。其中，以上靶细胞分别通过慢病毒转导方式，获得稳定表达萤火虫荧光素酶的靶细胞，因此样品中荧光素酶的活性可以反映靶细胞的数量。将CAR-T细胞和靶细胞共孵育培养。当靶细胞被CAR-T细胞杀伤时，荧光素酶会被释放并且很快失活(萤火虫荧光素酶半衰期约0.5h)。如果靶细胞没有被CAR-T细胞杀伤或者抑制，随着靶细胞的扩增和荧光素酶的持续表达，将会产生更多的荧光素酶。因此，可以通过荧光素酶的活性来检测CAR-T对靶细胞的杀伤情况。
体外细胞杀伤简要实验步骤：
1)将上述靶细胞分别在室温下以500g离心5min，弃上清后，用1640+10％FBS培养基重悬为1x10 5个细胞/mL；在96孔板的每孔中分别加入100μL靶细胞；
2)根据待测CAR-T样品的CAR阳性率和效靶比，分别在96孔板的每孔中加入100μL CAR-T细胞，并和靶细胞混匀；然后放入二氧化碳培养箱中孵育培养24h；
3)使用荧光素酶检测试剂盒分别检测每孔样品中的荧光素酶活性。
主要样品和试剂：
靶细胞U266-luc MM1.s-luc 8226-luc和Raji-luc CCRF-luc；
Steady-Glo Luciferase Assay System,Promega,Cat.No.E2520。
实验结果：
将CAR-T细胞样品和固定数量的靶细胞(1x10 4个)按照不同效靶比(E:T)混合后，共同孵育24h，然后检测样品中的荧光素酶活性(RLU)。由于荧光素酶活性可以反映靶细胞在样品中的数量，通过样品中荧光素酶活性的变化，可以得到CAR-T细胞对靶细胞的杀伤/抑制能力。荧光素酶活性读数(RLU)越低，靶细胞被杀伤的越多。
如图3A-3C所示，所有CAR-T细胞样品，均能有效杀伤GPRC5D阳性靶细胞，且与阴性靶细胞共同孵育时都无明显杀伤。其中克隆18，39，41的杀伤在8226-luc和U266-luc上均高于Benchmark 1。克隆46,47,14,8和Benchmark 1杀伤能力差不多,克隆10的杀伤较弱。
通过CD107a脱颗粒实验和体外细胞杀伤实验得到，克隆18,39,41在体外功能上都优于benchmark 1，下面再进一步针对这三个克隆的CAR的细胞因子，体外增殖能力等做进一步验证和评价。
实施例4. CAR细胞上细胞因子表达测定
实验目的和原理
采用U266 MM1.s 8226作为GPRC5D的阳性靶细胞，Raji和CCRF细胞作为GPRC5D阴性靶细胞，将GPRC5D CAR-T细胞和靶细胞孵育，采用流式方法测定CAR细胞胞内的IFN-γ和TNFα表达。
简要实验步骤：
1)将待测的CAR-T细胞和靶细胞分别在室温下以300g离心5min，弃上清后，用1640培养基+10％FBS重悬为8x10 6个细胞/mL；
2)在96孔中，分别加入50μL待测的CAR-T细胞和50μL靶细胞，并混匀；
3)在每孔细胞中加入50μL monensin和brefeldin A的预混液(相当于各0.2μL/孔)，然后放入细胞培养箱中37℃,5％CO 2孵育4h；
4)孵育完成后，在4℃下300g离心5min后弃上清，用200μL PBS洗细胞2次；
5)用100μLPBS重悬细胞，并分别加入1μL BV421anti-human CD8和1μL APC anti-human EGFR抗体，混匀后在冰上避光孵育20min；200μL PBS洗两遍。
6)每孔加入100μL固定破膜液，4℃20min，用1x wash buffer洗涤两次。
7)加入PE-TNFα和PE-Cy7-INFγ各1.5μL)抗体的buffer 50μL，4℃20min，用1x wash buffer洗涤两次。用100μL的PBS重悬后，用流式细胞仪检测。
主要样品和试剂：
靶细胞U266-luc(GPRC5D+),8226-luc(GPRC5D+),MM.1S-luc(GPRC5D+)
Raji-luc(GPRC5D-),CCRF-luc(GPRC5D-)
胎牛血清(FBS),Gibco，Cat.No.10099141；
Monensin,BioLegend,Cat.No.420701；
PE/Cy7 mouse anti-human CD107a,BD,Cat.No.561348；
BV421 mouse anti-human CD8,BD,Cat.No.301036；
APC anti-human EGFR Antibody,Clone AY13,BioLegend,Cat.No.35290
PE anti-human TNF-αBiolegend,Cat.No.502909
PE-Cy7anti-human IFNγBiolegend,Cat.No.506578。
实验结果
在Flowjo软件中分析，散点图中选取活细胞门(P1)，去除细胞碎片；在P1门中的细胞，经过分析选取单个分散细胞门(P2)；然后，在P2门中进一步选取CD8阳性的细胞(P3)；最后，在P3门中，分析TNF-α抗体或IFN-γ染色呈阳性的细胞(即CAR阳性细胞) 中TNF-α/IFN-γ阳性的比例。分析结果如表2所示，GPRC5D CAR-T仅与GPRC5D阳性细胞共孵育后TNFα和IFN-γ的表达水平有明显升高，而与阴性的细胞系共孵育后无明显的TNFα和IFN-γ的表达水平升高。其中克隆号18、39、41的GPRC5D阳性细胞的TNFα和IFN-γ的表达水平较高，且均高于Benchmark 1。同时这三种克隆在各种阳性靶细胞上的TNFα和IFN-γ的表达较均匀，Benchmark 1在U266和H929刺激下这两种细胞因子分泌较低。
表2 CAR-T细胞与多种靶细胞(8226-luc、U266-luc、MM1.s-luc、H929-luc、CCRF-luc、Raji-luc)共孵育后，CAR-T细胞内的细胞因子IFNγ阳性细胞和TNFα细胞在CAR中的百分比。
Figure PCTCN2022080836-appb-000012
Figure PCTCN2022080836-appb-000013
实施例5. 反复刺激增殖实验
实验目的和原理：
采用丝裂霉素(Mitomycin)处理过的靶细胞8226与不同组别GPRC5D CAR-T细胞混合，进行多次刺激后将CAR-T细胞和靶细胞共孵育培养，从而确定不同CAR-T在被靶细胞持续多次刺激后的增殖能力。
实验简要实验步骤：
1)取8226 7×10 6细胞，300g，室温，离心5min；
2)完全培养基调整密度至0.2×10 6细胞/mL，加入5μL Mitomycin母液(1μg/μL)混匀后培养24h后待用。
3)取处理24h后8226-Mitomycin细胞，300g，离心换液，用PBS洗涤6次，CTS培养基重悬8226-Mitomycin细胞，并计数且调整密度至6×10 6细胞/mL，待用。
4)分别取3×10 5CAR-T细胞，转入24孔板中。每孔CAR-T加入8226-Mitomycin细胞50μL，使效靶比E:T＝1：1。用CTS完全培养基补液至培养终体积至500μL，混匀，37度，5％CO 2培养96h并计数，再次用Mitomycin处理靶细胞(CCRF-CEM)，重复以上步骤并绘制扩增曲线。
主要样品和试剂：
Mitomycin C,MCE,Cat.No.HY-113061；
靶细胞：8226；
实验结果：
如表3和图4所示，4组CAR-T细胞样品反复刺激后均可有效扩增，靶细胞刺激7次后，克隆号18，39，41CAR-T细胞扩增能力强于Benchmark1扩增。CAR-T细胞被靶细胞刺激后的增殖能力与患者长期预后密切相关，因此，CAR克隆2290(#18)、2436(#39)、2438(#41)被认为具有更强的长期增殖并清除肿瘤细胞的潜能。
表3多次靶细胞(8226)刺激后各种CAR-T的体外增殖情况
增殖倍数 第一轮刺激 第二轮刺激 第三轮刺激 第四轮刺激 第五轮刺激 第六轮刺激 第七轮刺激
2254(Benchmark1) 7.15 41.89 101.26 172.01 239.50 320.87 312.64
2290(#18) 6.66 31.30 70.12 157.76 356.70 698.61 1292.66
2436(#39) 10.12 51.17 146.44 329.99 594.86 1155.33 2045.32
2438(#41) 10.60 46.29 111.16 230.11 492.90 1114.22 1617.85
实施例6. 器官来源肿瘤细胞刺激CAR细胞脱颗粒的性能测定
CD107a是细胞内微囊泡的标志物，当负载有颗粒酶的微囊泡与细胞膜融合后，细胞膜上的CD107a会增加，当用莫能酶素(monesin，购自BioLegend)阻断其回收时，可以定量反映微囊泡释放的强度。当CAR-T受到靶细胞上靶抗原刺激后，会造成颗粒酶释放，并可通过流式检测CD107a的增加来判断靶细胞对T细胞的激活情况。
CD107a脱粒简要实验步骤：
1)将待测的CAR-T细胞和靶细胞分别在室温下以300g离心5min，弃上清后，用1640培养基+10％FBS重悬为2x10 6个细胞/mL；
2)在96孔板中，分别加入100μL待测的CAR-T细胞和100μL靶细胞，并混匀；
3)在每孔细胞中加入1.5μL PE/Cy7anti-human CD107a抗体和0.2μLmonensin，然后放入细胞培养箱中(37℃,5％CO 2孵育4h；
4)孵育完成后，在4℃下300g离心5min后弃上清，用200μL PBS洗细胞2次；
5)用100μLPBS重悬细胞，并分别加入1.5μL BV421anti-human CD8和1.5μL APC anti-human EGFR抗体，混匀后在冰上避光孵育20min；
6)孵育完成后，用200μL PBS洗细胞3次；用100μLPBS重悬后，用流式细胞仪检测。
主要样品和试剂：
靶细胞293T A375 HepG2 Nalm6 NCHI460 OVCAR-3 HCT116 Panc-1 MDA-MB-468 KATOIII
胎牛血清(FBS),Gibco，Cat.No.10099141；
Monensin,BioLegend,Cat.No.420701；
PE/Cy7 mouse anti-human CD107a,BD,Cat.No.561348；
BV421 mouse anti-human CD8,BD,Cat.No.301036；
APC anti-human EGFR Antibody,Clone AY13,BioLegend,Cat.No.35290。
实验结果：
通过慢病毒转导的方式获得CAR-T细胞，将该CAR-T细胞在体外培养9-12天后进行CD107a脱粒实验。待检测的CAR-T细胞和靶细胞、莫能酶素和CD107a抗体共同孵育4h，CAR-T细胞与靶细胞的细胞密度均为2×10 6个细胞/mL。然后用CD8抗体、EGFR抗体标记样品后，进行流式检测。在Flowjo软件中分析，散点图中选取活细胞门(P1)，去除细胞碎片；在P1门中的细胞，经过分析选取单个分散细胞门(P2)；然后，在P2门中进一步选取CD8阳性的细胞(P3)；最后，在P3门中，分析EGFR抗体染色呈阳性的细胞(即CAR阳性细胞)中CD107a阳性的比例。分析结果如图5所示，GPRC5D CAR-T仅与GPRC5D阳性细胞共孵育后脱颗粒水平有明显升高，而与器官来源肿瘤细胞的细胞系共孵育后无明显的脱颗粒水平升高。表明GPRC5D CAR-T细胞不易被正常的组织器官细胞激活，具有较高的安全性。
参考文献
1.Shah U A,Mailankody S.Emerging immunotherapies in multiple myeloma.BMJ(online),2020,370:m3176
2.Soekojo C Y,Ooi M,Mel S D,et al.Immunotherapy in Multiple Myeloma.Cells,2020,9(3):601.
3.Feng,D,Sun,J.Overview of anti-BCMA CAR-T immunotherapy for multiple myeloma and relapsed/refractory multiple myeloma.Scand J Immunol.2020；92:e12910.
4.Nikhil C.Munshi,Larry D.Anderson,Jr.,Nina Sha et al.Idecabtagene Vicleucel in Relapsed and Refractory Multiple Myeloma.N Engl J Med 2021；384:705-716
5.Smith EL,et al.GPRC5D is a target for the immunotherapy of multiple myeloma with rationally designed CAR T cells.Sci Transl Med.2019Mar.27；11(485)
6.Kodama T,Yu K,Nakai W,et al.Anti-GPRC5D/CD3 Bispecific T-Cell–Redirecting Antibody for the Treatment of Multiple Myeloma.Molecular Cancer Therapeutics,2019,18(9):molcanther.1216.2018.
7.https://www.mousephenotype.org/data/genes/MGI:1935037
8.Zheng,Y.J.,Nandakumar,K.S.,Cheng,K.Optimization of CAR-T Cell-Based Therapies Using Small-Molecule-Based Safety Switches,J.Med.Chem.2021,64,9577-9591.

Claims (31)

  1. 嵌合抗原受体(CAR)，其胞外抗原结合结构域包括一个或更多个靶向GPRC5D的抗体分子或其抗原结合片段，所述抗体分子的重链可变区的HCDR1、HCDR2和HCDR3选自如下组合之一：
    1)HCDR1的序列为GGSFSGYY(SEQ ID NO：1)；
    HCDR2的序列为INHSGST(SEQ ID NO：2)；
    HCDR3的序列为ARARRYGGRTRFDP(SEQ ID NO：3)；
    2)HCDR1的序列为GFIFSSYG(SEQ ID NO：4)；
    HCDR2的序列为ISSSGDYT(SEQ ID NO：5)；
    HCDR3的序列为ARMSFRRYDH(SEQ ID NO：6)；以及
    3)HCDR1的序列为GFSFSGYI(SEQ ID NO：7)；
    HCDR2的序列为TSSSGTET(SEQ ID NO：8)；
    HCDR3的序列为ARYYSKYGRSYHVDS(SEQ ID NO：9)。
  2. 如权利要求1所述的CAR，其中所述胞外抗原结合结构域包括串联连接的两个所述抗体分子或其抗原结合片段，优选地，两个所述抗体分子或其抗原结合片段通过连接肽相连。
  3. 如权利要求2所述的CAR，其中所述两个所述抗体分子或其抗原结合片段相同或不同。
  4. 如权利要求1-3任一项所述的CAR，其中所述抗体分子为单域抗体，优选地，所述抗体分子为全人源单域抗体。
  5. 如权利要求1-4任一项所述的CAR，其中所述重链可变区包括SEQ ID NO：10、11或12所示的氨基酸序列；或者所述重链可变区包括与SEQ ID NO：10、11或12所示序列有至少90％、91％、92％、93％、94％、95％、96％、97％、98％、或99％序列一致性的氨基酸序列并且能够特异性结合GPRC5D。
  6. 如权利要求1-5任一项所述的CAR，其中所述CAR从N端到C端依次包括所述胞外抗原结合结构域、跨膜结构域和胞内信号传导结构域。
  7. 如权利要求1-6任一项所述的CAR，其中所述CAR在N末端还包括信号肽。
  8. 如权利要求1-7任一项所述的CAR，其中所述信号肽包括SEQ ID NO：17所示的氨基酸序列。
  9. 如权利要求1-8任一项所述的CAR，其中所述CAR在所述胞外抗原结合结构域和所述跨膜结构域之间还包括铰链区。
  10. 如权利要求1-9任一项所述的CAR，其中所述铰链区包括SEQ ID NO：19所示的氨基酸序列。
  11. 如权利要求1-10任一项所述的CAR，其中所述跨膜区包括SEQ ID NO：21所示的氨基酸序列。
  12. 如权利要求1-11任一项所述的CAR，其中所述胞内信号传导结构域包括4-1BB胞内结构域和CD3ζ胞内结构域。
  13. 如权利要求1-12所述的CAR，其中所述4-1BB胞内结构域包括SEQ ID NO：23所示的氨基酸序列，和/或所述CD3ζ胞内结构域包括SEQ ID NO：25所示的氨基酸序列。
  14. 如权利要求1-13任一项所述的CAR，其中所述CAR具有SEQ ID NO：35、37或39所示的氨基酸序列；或者所述CAR的氨基酸序列与SEQ ID NO：35、37或39所示序列有至少90％、91％、92％、93％、94％、95％、96％、97％、98％、或99％的序列一致性。
  15. 如权利要求1-14任一项所述的CAR，其中所述CAR的末端还连接有用于CAR-T细胞清除的安全开关；优选地，在所述CAR的C末端连接有安全开关；更优选地，所述安全开关包括截短形式的EGFR分子(tEGFR)或具有自杀能力的融合蛋白。
  16. 如权利要求1-15任一项所述的CAR，其中所述CAR的C末端还连接有自裂解多肽和tEGFR；优选地，在tEGFR的N末端还连接有CSF2RA信号肽；更优选地，所述tEGFR包括SEQ ID NO：31所示的氨基酸序列，和/或所述CSF2RA信号肽包括SEQ ID NO：29所示的氨基酸序列。
  17. 如权利要求1-16任一项所述的CAR，其中所述安全开关通过自裂解多肽与CAR相连，优选地，所述自裂解多肽包括SEQ ID NO：27或33所示的氨基酸序列。
  18. 如权利要求1-17任一项所述的CAR，其中所述CAR为双特异性CAR，所述胞外抗原结合结构域还包括靶向第二靶点的第二抗体分子或其抗原结合片段，所述第二靶点选自CD3、BCMA或其组合。
  19. 分离的核酸分子，其编码权利要求1-18任一项所述的CAR。
  20. 如权利要求19所述的核酸分子，其中所述核酸分子包括SEQ ID NO：13、14、15、16、18、20、22、24、26、28、30、32、34、36和38任一项所示的核苷酸序列。
  21. 表达载体，其包括权利要求20所述的核酸分子。
  22. 如权利要求21所述的表达载体，其选自质粒、逆转录病毒载体和慢病毒载体。
  23. 宿主细胞，其包括权利要求1-18任一项所述的CAR、权利要求19或20所述的核酸分子或权利要求21或22所述的表达载体。
  24. 一种工程化免疫细胞，其表达权利要求1-18任一项所述的CAR。
  25. 如权利要求24所述的工程化免疫细胞，为免疫细胞，优选地，为自体免疫细胞或异体免疫细胞，更优选地，所述免疫细胞为T细胞或NK细胞。
  26. 药物组合物，其包括权利要求23-25任一项所述的细胞以及药学上可接受的载体。
  27. 权利要求1-18中任一项所述的CAR、权利要求19或20所述的核酸分子、权利要求21或22所述的表达载体、或权利要求23-25任一项所述的细胞在制备用于预防或治疗GPRC5D相关疾病的药物中的用途。
  28. 如权利要求27所述的用途，其中所述GPRC5D相关疾病为癌症或自身免疫疾病，所述癌症优选浆细胞恶性肿瘤疾病，例如多发性骨髓瘤；或者B细胞恶性疾病，例如霍奇金淋巴瘤或非霍奇金淋巴瘤。
  29. 预防或治疗GPRC5D相关疾病的方法，包括以治疗有效量的权利要求23-25任一项所述的细胞或权利要求26所述的药物组合物向有需要的受试者给药。
  30. 如权利要求29所述的方法，还包括以EGFR抗体向所述受试者给药来抑制所述免疫效应细胞或所述药物组合物的活性。
  31. 如权利要求29或30所述的方法，其中所述GPRC5D相关疾病为癌症或自身免疫疾病，所述癌症优选浆细胞恶性肿瘤疾病，例如多发性骨髓瘤；或者B细胞恶性疾病，例如霍奇金淋巴瘤或非霍奇金淋巴瘤。
PCT/CN2022/080836 2021-12-21 2022-03-15 靶向gprc5d的全人源嵌合抗原受体(car)及其应用 WO2023173272A1 (zh)

Priority Applications (7)

Application Number Priority Date Filing Date Title
PCT/CN2022/080836 WO2023173272A1 (zh) 2022-03-15 2022-03-15 靶向gprc5d的全人源嵌合抗原受体(car)及其应用
CA3241997A CA3241997A1 (en) 2021-12-21 2022-12-21 Fully human antibody targeting gprc5d and chimeric antigen receptor (car) and use thereof
IL313798A IL313798A (en) 2021-12-21 2022-12-21 Fully human antibody and Chimeric Antigen Receptor (CAR) against GPRC5D and their use
KR1020247024554A KR20240125026A (ko) 2021-12-21 2022-12-21 Gprc5d를 표적으로 하는 완전 인간 항체와 키메라 항원 수용체(car) 및 이의 용도
PCT/CN2022/140769 WO2023116782A1 (zh) 2021-12-21 2022-12-21 靶向gprc5d的全人源抗体和嵌合抗原受体(car)及其应用
CN202280083287.1A CN118451177A (zh) 2021-12-21 2022-12-21 靶向gprc5d的全人源抗体和嵌合抗原受体(car)及其应用
AU2022418639A AU2022418639A1 (en) 2021-12-21 2022-12-21 Fully human antibody targeting gprc5d and chimeric antigen receptor (car) and use thereof

Applications Claiming Priority (1)

Application Number Priority Date Filing Date Title
PCT/CN2022/080836 WO2023173272A1 (zh) 2022-03-15 2022-03-15 靶向gprc5d的全人源嵌合抗原受体(car)及其应用

Publications (1)

Publication Number Publication Date
WO2023173272A1 true WO2023173272A1 (zh) 2023-09-21

Family

ID=88022046

Family Applications (1)

Application Number Title Priority Date Filing Date
PCT/CN2022/080836 WO2023173272A1 (zh) 2021-12-21 2022-03-15 靶向gprc5d的全人源嵌合抗原受体(car)及其应用

Country Status (1)

Country Link
WO (1) WO2023173272A1 (zh)

Cited By (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN118108846A (zh) * 2024-02-04 2024-05-31 四川大学华西医院 靶向GPRC5D同种异体CAR-γδT细胞的制备及应用

Citations (6)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN109715667A (zh) * 2016-07-20 2019-05-03 詹森药业有限公司 抗-gprc5d抗体、结合gprc5d和cd3的双特异性抗原结合分子及其用途
CN110462038A (zh) * 2017-02-07 2019-11-15 第一三共株式会社 抗gprc5d抗体和包含所述抗体的分子
CN111788231A (zh) * 2018-02-09 2020-10-16 豪夫迈·罗氏有限公司 结合gprc5d的抗体
WO2021018925A1 (en) * 2019-07-31 2021-02-04 F. Hoffmann-La Roche Ag Antibodies binding to gprc5d
WO2021113780A1 (en) * 2019-12-06 2021-06-10 Juno Therapeutics, Inc. Anti-idiotypic antibodies to gprc5d-targeted binding domains and related compositions and methods
CN113597433A (zh) * 2019-01-18 2021-11-02 詹森生物科技公司 Gprc5d嵌合抗原受体以及表达这些受体的细胞

Patent Citations (6)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN109715667A (zh) * 2016-07-20 2019-05-03 詹森药业有限公司 抗-gprc5d抗体、结合gprc5d和cd3的双特异性抗原结合分子及其用途
CN110462038A (zh) * 2017-02-07 2019-11-15 第一三共株式会社 抗gprc5d抗体和包含所述抗体的分子
CN111788231A (zh) * 2018-02-09 2020-10-16 豪夫迈·罗氏有限公司 结合gprc5d的抗体
CN113597433A (zh) * 2019-01-18 2021-11-02 詹森生物科技公司 Gprc5d嵌合抗原受体以及表达这些受体的细胞
WO2021018925A1 (en) * 2019-07-31 2021-02-04 F. Hoffmann-La Roche Ag Antibodies binding to gprc5d
WO2021113780A1 (en) * 2019-12-06 2021-06-10 Juno Therapeutics, Inc. Anti-idiotypic antibodies to gprc5d-targeted binding domains and related compositions and methods

Cited By (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN118108846A (zh) * 2024-02-04 2024-05-31 四川大学华西医院 靶向GPRC5D同种异体CAR-γδT细胞的制备及应用

Similar Documents

Publication Publication Date Title
US10233258B2 (en) Bispecific binding proteins that bind CD40 and mesothelin
RU2746754C2 (ru) Индуцирующее повреждение клеток терапевтическое лекарственное средство, предназначенное для противораковой терапии
US12018077B2 (en) Methods of treatments using antigen-binding proteins targeting CD56
AU2015357543B2 (en) Chimeric antigen receptors targeting Fc Receptor-like 5 and uses thereof
US20240173352A1 (en) Cell-surface receptors responsive to loss of heterozygosity
CA3030837A1 (en) Treatment and prevention of cytokine release syndrome using a chimeric antigen receptor in combination with a kinase inhibitor
JP2020513839A (ja) Ｔｉｍ−１を標的とするキメラ抗原受容体
CN113412117A (zh) 嵌合抗原和t细胞受体及使用的方法
US20210393692A1 (en) Compositions and methods for adoptive cell therapy for cancer
TW202023580A (zh) 使用靶特異性融合蛋白進行ｔｃｒ再程式化之組合物及方法
CN111848809A (zh) 靶向Claudin18.2的CAR分子、其修饰的免疫细胞及用途
JP2022530301A (ja) Ｃｄ３抗原結合性断片及びその使用
WO2019047885A1 (en) IMMUNOCONJUGATES COMPRISING ALPHA REGULATORY SIGNAL PROTEIN
WO2023116782A1 (zh) 靶向gprc5d的全人源抗体和嵌合抗原受体(car)及其应用
CA3132462A1 (en) Therapeutic antigen binding proteins specific for cd93 and methods of use thereof
WO2023093811A1 (zh) 分子开关调控型嵌合抗原受体细胞和抗体的组合及其应用
WO2023056429A1 (en) Anti-nmdar2b antibodies, antibody-drug conjugates, and chimeric antigen receptors, and compositions and methods of use
WO2023173272A1 (zh) 靶向gprc5d的全人源嵌合抗原受体(car)及其应用
WO2023104099A1 (zh) 靶向bcma的p329g抗体及其与嵌合抗原受体细胞的组合和应用
WO2023030539A1 (en) Anti-gpc3 chimeric antigen receptor and methods of use thereof
US20220267420A1 (en) Foxp3 targeting agent compositions and methods of use for adoptive cell therapy
CN114685659A (zh) Cd22特异性人源化抗体及利用其的嵌合抗原受体
CN114933654B (zh) 靶向cd123的抗体、嵌合抗原受体及其用途
WO2022253240A1 (en) Antibodies targeting afp peptide/mhc complexes and uses thereof
WO2023246574A1 (zh) 靶向gpc3的抗体及其用途

Legal Events

Date Code Title Description
121 Ep: the epo has been informed by wipo that ep was designated in this application

Ref document number: 22931312

Country of ref document: EP

Kind code of ref document: A1